Methods of detecting tumor cells

ABSTRACT

In one aspect, methods of detecting tumor cells are described herein. In some embodiments, a method of detecting tumor cells comprises providing a device, the device comprising a substrate surface and a plurality of first aptamer probes attached to the substrate surface. The method further comprises contacting a plurality of cells with the plurality of first aptamer probes attached to the substrate surface; adhering one or more of the plurality of cells to the substrate surface; and measuring a non-uniformity parameter, a Hausdorff distance, a change in the number of pseudopods, and/or a shape of at least one adhered cell. In some cases, the method further comprises using the non-uniformity parameter, Hausdorff distance, change in the number of pseudopods, and/or shape of the adhered cell to identify the adhered cell as a tumor cell or a non-tumor cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application under 35 U.S.C. § 120 ofU.S. patent application Ser. No. 14/323,840, filed Jul. 3, 2014, whichis a continuation-in-part of U.S. patent application Ser. No.13/081,319, filed Apr. 6, 2011, which claims priority pursuant to 35U.S.C. § 119 to U.S. Patent Application Ser. No. 61/321,770, filed Apr.7, 2010, each of which is hereby incorporated by reference in itsentirety.

The sequence listing titled “Sequence Listing” having a file size of2,204 bytes and filed herewith is incorporated herein by reference as iffully set forth.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CAREER awardcontract ECCS 0845669 awarded by the National Science Foundation (NSF).The government has certain rights in the invention.

FIELD

The invention relates generally to devices, systems, and methods fordetecting hybridization and/or morphology of proteins and/or cells,including cancer cells, using surface-tethered aptamer probes, withoutthe use of labeling or target modification.

BACKGROUND

Antibodies are commonly used to functionalize nano-devices andnano-objects for detection of specific biomarkers. Such antibody-basedmolecular recognition has limited capability for field-deployable orpoint-of-care modalities, as antibodies need a certain range oftemperatures, humidity, and solution conditions to retain theirstructure. In terms of the solution conditions, an important parameteris the need for low ionic strength. Low ionic strength of the buffersolutions is needed to overcome surface Debye screening, but it alsoresults in weak interactions between the surface probe and solutiontarget.

There are other means of protein detection, including amperometric andoptical detection, such as those employing microarray technology.Electrical detection methods include capacitive, impedometric, andvoltametric detection. In the case of optical detection, a fluorescenttag is usually attached to the DNA or the protein and the change influorescent intensity is measured after binding. However, the tagging ofthe DNA or protein molecule can change the thermodynamic properties ofthe molecular interactions of DNA and, in some cases, unnaturallystabilize or destabilize the DNA double-strand of the DNA or protein andchange the melting temperature significantly. Additionally, expensivefluorescent microscopes are needed to visualize the data andnormalization of the data to references remains problematic. Further,the hybridization of the probe and target molecules is adiffusion-limited process requiring long-incubation times as the targetmolecules must travel to the arrayed probes on the surface of the chip.The fluorophores are also known to have great effect on the stability ofthe duplexes as a function of the sequence itself. In addition,fluorescent dyes photobleach, quench statically, or interact with eachother, so the microarray technologists need to have very detailedknowledge about the limitations of the optics, reagents used, and thesample interactions.

Thus, there is a need for biosensors for proteins and cells, includingcancer cells, that integrate sensing, characterization, comparativeanalysis and decision making all on-board a single chip, whilesustaining or increasing sensitivity and specificity. The invention isdirected to these, as well as other, important ends.

SUMMARY

The invention provides a nanotechnology-based low-power, rapid,inexpensive, recyclable, and sensitive electrical detection device,system, and method of low concentrations of proteins and cells with noexternal sample preparation or labeling or other chemical modificationof the sample. The biosensors of the invention may be used in widevariety of applications requiring sensitive protein and cell detection,including, but not limited to, cancer cell detection, forensics, earlydisease detection, disease progression monitoring (such as in responseto therapy and/or medicinal agents), legal matters (such as paternityand criminal proceedings), defensive biohazard detection, andimmigration issues (such as establishing blood relationships). Thebiosensors of the invention are useful in further enabling “personalizedmedicine,” where drugs are designed according to each individual'sgenetic make-up.

The invention involves a number of features: use of nanoimprintlithography-based techniques to make chips with arrays of nanogapelectrodes and embedded heaters for on-chip sample treatment, forrecycling of the chip for next batch of targets, and for temperaturegradient focusing of the target proteins and cells; use of surface boundlinear double-stranded nucleic acid aptamer or hairpin loop nucleic acidprobes that are capable of distinguishing between solution phasespecific binding and single-base mismatched sequences; use of ananoparticle conjugated to a short sequence of nucleic acids as adetector to quantify the level of hybridization between probe and targetDNA molecules; and use of low-power printed circuit board electronics tointerrogate the mass fabricated interaction sites and electrical sensingof the protein-nucleic acid and cell-nucleic acid hybridization.

In one embodiment, the invention is directed to devices, comprising: athermally responsive, electrically insulating substrate; at least oneheating element; and a first detecting unit, comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof first aptamer probes attached to said substrate in said nanogap.

In other embodiments, the invention is directed to devices, wherein saidfirst aptamer probes are double-stranded nucleic acids and have the samenucleic acid sequence.

In yet other embodiments, the invention is directed to devices, whereinsaid first aptamer probes comprise: a first nucleic acid portion in ahairpin loop formation; wherein said first nucleic acid portion comprisea spacer, a loop, and a stem region, said stem region beingdouble-stranded; and a second nucleic acid portion in a linearformation; wherein said second nucleic acid portion is single strandedand is attached to said substrate; and wherein said second nucleic acidportion is complementary to at least a portion of said spacer in saidfirst nucleic acid portion; and wherein each of said first aptamerprobes has the same nucleic acid sequence.

In yet another embodiment, the invention is directed to systems,comprising: a device described herein; and an electrical reading devicefor interrogating said device; wherein said electrical reading device isoptionally portable.

In one embodiment, the invention is directed to methods for detectinghybridization of a target, comprising: providing a device, comprising: athermally responsive, electrically insulating substrate; at least oneheating element; and a first detecting unit, comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof first aptamer probes attached to said substrate in said nanogap;providing a solution comprising said target under hybridizingconditions; wherein said target is a protein or a cell; and wherein saidtarget hybridizes at least some of said first aptamer probes; applying avoltage drop across said electrodes; and measuring a change inconductivity, resistivity, capacitance, or impedance across saidelectrodes at known locations to determine perfect complementarity ofsaid target to said first aptamer probes.

In another aspect, methods of detecting tumor cells are describedherein. In some embodiments, a method of detecting tumor cells comprisescontacting a plurality of cells with a substrate of a device describedherein. Any device described herein may be used. In some cases, thedevice comprises a substrate surface and a plurality of first aptamerprobes attached to the substrate surface. Moreover, a method ofdetecting tumor cells described herein can further comprise contacting aplurality of cells with the plurality of first aptamer probes attachedto the substrate surface; adhering one or more of the plurality of cellsto the substrate surface; and measuring a non-uniformity parameter, aHausdorff distance, a change in the number of pseudopods, and/or a shapeof at least one adhered cell. Moreover, in some instances, such a methodfurther comprises using the non-uniformity parameter, Hausdorffdistance, change in the number of pseudopods, and/or shape of theadhered cell to identify the adhered cell as a tumor cell or a non-tumorcell. Additionally, in some embodiments, the plurality of first aptamerprobes forms a uniform or substantially uniform coating on the substratesurface. Further, the plurality of first aptamer probes can comprise oneor more anti-EGFR aptamers.

In some embodiments of methods described herein, the plurality of cellscomprises tumor cells, including circulating tumor cells (CTCs).Moreover, in some cases, the plurality of cells comprises a mixture oftumor cells and non-tumor cells. In addition, in some instances,contacting a plurality of cells with the plurality of first aptamerprobes comprises contacting a bodily fluid such as urine, blood, orsaliva with the plurality of first aptamer probes. Contacting aplurality of cells with the plurality of first aptamer probes can alsocomprise contacting an operative specimen, pleural fluid, ascites,spinal fluid, benign tissue, or suspected malignant tissue with theplurality of first aptamer probes.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate embodiments of the invention andtogether with the description serve to explain the principles of theinvention.

FIG. 1A is a three-dimensional view of metal nanoplates as heatingelements 2 a, 2 b embedded on a silicon dioxide chip 1. FIG. 1B is aside view of the metal nanoplates as heating elements 2 a, 2 b embeddedon the silicon dioxide chip 1, shown in FIG. 1A.

FIG. 2 is a side view of metal nanoplates as heating elements 2 a, 2 bembedded on silicon dioxide chip 1 showing typical temperature gradients3 a, 3 b for each heating element 2 a, 2 b.

FIG. 3A is a top view of metal nanoplates as heating elements 2 a, 2 bembedded on a silicon dioxide chip 1 with nanogap electrodes 5 a, 5 busing a temperature gradient to focus or move target molecules 10 towardthe nanogap electrodes 5 a, 5 b.

FIG. 3B is a side view of metal nanoplates as heating elements 2 a, 2 bembedded on a silicon dioxide chip 1 with nanogap electrodes 5 a, 5 bshowing the movement of the buffer solution containing theoligonucleotide target molecules 10 toward the nanogap electrodes 5 a, 5b.

FIG. 4A is a schematic view of metal nanoplates as heating elements 2 a,2 b embedded on a silicon dioxide chip 1 with a set of electrodes 5 a, 5b with a nanogap 6.

FIG. 4B is an expanded side view of metal nanoplates as heating elements2 a, 2 b embedded on a silicon dioxide chip 1 with a set of electrodes 5a, 5 b with a nanogap 6 shown in FIG. 4A, but also showing the attachedhairpin loop aptamer probes 8.

FIG. 5A is a top view of one embodiment showing two bus lines 9 a, 9 bused to address the individual set of electrodes 5 a, 5 b with a nanogap6 fabricated with nanoimprint lithography. Arrows indicate the extensionof similar structures to cover the whole chip area.

FIG. 5B is a blown up schematic (not to scale) of FIG. 5A showing theindividual set of electrodes 5 a, 5 b with a nanogap 6, one electrodeaddressing/measuring bus 9 a covered with silicon nitride and the otherelectrode addressing/measuring bus 9 b. Oligonucleotide probe molecules(not shown) are attached to the chip in the nanogap 6 between theelectrodes 5 a, 5 b.

FIG. 6 is a schematic diagram showing one embodiment of the inventionwhere a chip and a cover block with microchannels for fluid handling areshown.

FIG. 7 shows the gold electrodes with a nanogap prior to attachment ofthe hairpin loop probes of one embodiment of the invention.

FIG. 8 shows a micrograph of the device of one embodiment of theinvention after two-step photolithography and gold lift-off process.

FIG. 9 shows a micrograph of a break-junction chip of one embodiment ofthe invention after FIB at different process conditions (a) at 30 KVapplied voltage, 1 pA milling current and 30 secs of scratching time (b)at 30 KV applied voltage, 20 pA milling current and 120 secs ofscratching time.

FIG. 10 shows an SEM micrograph of a representative break junction afterelectromigration of one embodiment of the invention.

FIG. 11 shows I-V data comparing current through the junction before andafter the break.

FIG. 12 compares G-V data for a specific break junction before and afterthe capture of EGFR protein using anti-EGFR aptamer represents aremarkable increase in electrical conductance.

FIG. 13 comparises G-V data for a control break junction before andafter the same surface functionlization minus anti-EGFR aptamers showsno change in conducatnce depicting no capture of EGFR proteins.

FIG. 14 shows the crystal structure of the extra-cellular region ofhuman EGFR. The measurements (in angstroms) represent the widest pointsof the structure: (a) Front view, (b) Bottom view. Images are made withPyMOL 1.2.8 (evaluation version).

FIG. 15 is a fluorescence image of dsDNA stained with Acridine Orange onthe surface of CMOS chip. Acridine Orange fluoresces green when itinteracts with dsDNA.

FIG. 16 shows the results of EMSA PAGE gel. The first 4 lanes (from L toR) 8.4 pmole, 2.8 pmole, 0.84 pmole, and 0.28 pmole protein,respectively, of R2Bm protein bound to 1 pM of dsDNA. The last two lanesare dsDNA and ssDNA, respectively, in the absence of protein.

FIG. 17 shows Sypro stain intensity measurements on chips with thefollowing surface modifications: Chip 1 and 2: DNA & protein attached onchip; Chip 3: Only DNA immobilized on chip; Chip 4: Only protein on chip(No DNA); Chip 5: Only APTMS modification on chip surface; Chip 6:Piranha cleaned chip surface (no biomolecule). These results areaverages of 10 chips (n=10).

FIG. 18 is a schematic diagram of the attachment of dsDNA and protein onthe proteomic biochip on one embodiment of the invention.

FIG. 19 shows I-V measurements comparing current measured between metalnano-electrodes. Control data is red triangles with no surface bounddsDNA and protein molecules. The blue stars show I-V data for the chipwith the DNA and the protein immobilized on its surface.

FIG. 20 is an SEM micrograph of the pads. Scale bar: 2 μm.

FIG. 21 shows Anti-EGFR aptamer binding to the cultured mouse derivedtumor cell. The RNA aptamer was annealed to 6-FAM modified DNA captureprobe. The RNA aptamer-DNA probe complex was allowed to interact andbind to mouse-derived tumor cells at 37° C. for 30 minutes in 5% CO₂.After binding, the cells were washed with 1×PBS three times. (a) showsschematic depicting mouse derived cell bound with aptamer complex; (b)shows overlaid fluorescent and DIC images. The green fluorescent showsthe cell-bound aptamer molecules; (c) shows average fluorescenceintensity of each group.

FIG. 22 is a schematic diagram showing steps of experiments. Theamine-modified DNA capture probes were first immobilized on the glasssubstrates. After hybridization with 1 μM anti-EGFR RNA aptamer at 37°C. for 2 hours, substrates were incubated with tumor cells at 37° C. for30 minutes. After incubation the substrates were washed with 1×PBS for 8minutes.

FIG. 23 shows the density and size ranges of captured cells. Deviceswere incubated with mouse derived tumor cells and washed with 1×PBS. (a)Plot shows average tumor cell density on 12 anti-EGFR aptamer substrates(Avg: 392 cells/m , Max: 831 cells/mm², Min: 284 cells/mm², S.D: 143.3)and on 12 control substrates with mutant aptamer (Avg: 7 cells/mm², Max:11 cells/mm², Min: 0 cells/mm², S.D: 2.8), *P<0.01; (b) Plot showsdistribution of the diameters of tumor cells on 12 anti-EGFR aptamersubstrates; (c) and (d) shows representative tumor cells on mutantaptamer (c) and anti-EGFR aptamer grafted surfaces (d).

FIG. 24 shows human GBM cells on the substrates. Devices were incubatedwith hGBM and washed with PBS. The hGBM cells captured on (a) theanti-EGFR aptamer substrate and (b) mutant aptamer control substrate.(c) Plot shows average hGBM cells density on 12 anti-EGFR aptamersubstrates (Avg: 117 cells/mm², Max: 228 cells/mm², Min: 56 cells/mm²,S.D: 44.4) and on 12 control substrates with mutant aptamer (Avg: 4cells/mm², Max: 13 cells/mm², Min: 0 cells/mm², S.D: 4.1) (*P<0.01).

FIG. 25 shows the hGBM cells and fibroblast on the substrate surfaces.Substrates were incubated with mixture of hGBM and fibroblast and washedwith PBS. (a) and (b) are DIC and fluorescent images respectively fromsame position. The circles in (a) indicate a few fibroblasts that werecaptured and cannot be seen in (b).

FIG. 26 shows the changes in shapes of mouse-derived tumor cells. (a)and (c) were taken 3 minutes after seeding the cells on the anti-EGFRand mutant aptamer substrates, respectively. (c) and (d) were taken 30minutes later on the anti-EGFR and mutant aptamer substrates,respectively. (a) to (c) shows the change of cell shapes on theanti-EGFR aptamer grafted surface in 30 minutes. (b) to (d) shows nochanges in cell shape cells on mutant aptamer substrates.

FIG. 27 shows SEM micrographs of NaOH treated and untreated PLGA surface(the inset is untreated one). The micrographs show that nano-texturedPLGA has a higher degree of surface roughness.

FIG. 28 shows PLGA surface roughness increased after NaOH etching. TheAFM micrographs (3×3 μm²) of (A) untreated PLGA; (B) after 10 N NaOHetching for 1 hour. The Surface roughness increased from 22 nm onuntreated PLGA surface to 310 nm on nanostructured PLGA surface.

FIG. 29 shows human GBM cells on the anti-EGFR and mutant aptamermodified glass, PDMS and nano-textured PDMS substrates. Substrates wereincubated with hGBM and washed with PBS. The hGBM cells densities (permm²) on the anti-EGFR aptamer modified (A) glass, (C) PDMS and (E)nano-textured PDMS substrates are 79.3 (S.D.: 11.5), 37.4 (S.D.: 10.1),and 149.6 (S.D.: 12.2) respectively; the cell densities on the mutantaptamer modified (B) glass, (D) PDMS and (F) nano-textured PDMSsubstrates are 2.2 (S.D.: 1.2), 0.6 (S.D.: 0.8), and 25.6 (S.D.: 6.2)respectively; (*P<0.05). (G) Plot shows average hGBM cells density oneach type of substrate. The table in the inset depicts actual numbers ofthe plot.

FIG. 30 shows SEM micrographs of captured tumor cell on (A) PDMS and (B)nano-textured PDMS substrate. Images show that cell can firmly attach onthe rough surface which mimic the basement membrane structure. The scalebar is 1 μm. Cells were fixed in 4% paraformaldehyde for 3 h, and thenthe substrates were immersed into 20%, 30%, 50%, 70%, 85%, 95% and 100%(v/v) ethanol concentration gradient solution (15 min in eachconcentration). All substrates were lyophilized overnight.

FIG. 31 shows the hGBM cells and fibroblast on the nano-textured PDMSsubstrates. Substrates were incubated with mixture of hGBM andfibroblast and washed with PBS. (A) and (B) are DIC and fluorescentimages respectively from same position. The circles in (A) indicate afew fibroblasts that were captured and cannot be seen in (B).

FIG. 32 illustrates steps of a method of detecting tumor cells accordingto one embodiment described herein.

FIGS. 33-36 each illustrates a step of a method of detecting tumor cellsaccording to one embodiment described herein.

FIG. 37 illustrates comparative data regarding a method of detectingtumor cells according to one embodiment described herein.

FIGS. 38 and 39 each illustrates data regarding a method of detectingtumor cells according to some embodiments described herein.

DETAILED DESCRIPTION

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the drawings and theexamples. This invention may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein. In addition and as will be appreciated by one of skill inthe art, the invention may be embodied as a product, method, system orprocess.

In addition, all ranges disclosed herein are to be understood toencompass any and all subranges subsumed therein. For example, a statedrange of “1.0 to 10.0” should be considered to include any and allsubranges beginning with a minimum value of 1.0 or more and ending witha maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or3.6 to 7.9.

All ranges disclosed herein are also to be considered to include the endpoints of the range, unless expressly stated otherwise. For example, arange of “between 5 and 10” should generally be considered to includethe end points 5 and 10.

Further, when the phrase “up to” is used in connection with an amount orquantity, it is to be understood that the amount is at least adetectable amount or quantity. For example, a material present in anamount “up to” a specified amount can be present from a detectableamount and up to and including the specified amount.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. The following definitions areprovided for the full understanding of terms used in this specification.

As used herein, the article “a,” “an,” and “the” means “at least one,”unless the context in which the article is used clearly indicatesotherwise.

As used herein, the term “nucleic acid” as used herein means a polymercomposed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.

As used herein, the terms “ribonucleic acid” and “RNA” as used hereinmean a polymer composed of ribonucleotides.

As used herein, the terms “deoxyribonucleic acid” and “DNA” as usedherein mean a polymer composed of deoxyribonucleotides.

As used herein, the term “oligonucleotide” denotes single- ordouble-stranded nucleotide multimers of from about 2 to up to about 100nucleotides in length.

Suitable oligonucleotides may be prepared by the phosphoramidite methoddescribed by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862(1981), or by the triester method according to Matteucci, et al., J. Am.Chem. Soc., 103:3185 (1981), both incorporated herein by reference, orby other chemical methods using either a commercial automatedoligonucleotide synthesizer or VLSIPS™. technology. Whenoligonucleotides are referred to as “double-stranded,” it is understoodby those of skill in the art that a pair of oligonucleotides exist in ahydrogen-bonded, helical array typically associated with, for example,DNA. In addition to the 100% complementary form of double-strandedoligonucleotides, the term “double-stranded,” as used herein is alsomeant to refer to those forms which include such structural features asbulges and loops, described more fully in such biochemistry texts asStryer, Biochemistry, Third Ed., (1988), incorporated herein byreference for all purposes.

As used herein, the term “cDNA” means a complementary DNA molecule madeas a copy of mRNA amplified using PCR for deposition on arrays. cDNAscan range from about 100 bp to about 8,000 bp, where average cDNAs aretypically 1 to 2 kb in length.

As used herein, the term “array” means a substrate having a plurality ofbinding agents (probes) stably attached to its surface, where thebinding agents (probes) are arranged in a spatially defined andphysically addressable manner across the surface of the substrate in anyof a number of different patterns. Generally, at least two of theplurality of binding agents (probes) is different.

As used herein, the term “complementary” refers to the topologicalcompatibility or matching together of interacting surfaces of a probemolecule and its target. Thus, the target and its probe can be describedas complementary, and furthermore, the contact surface characteristicsare complementary to each other.

As used herein, the term “bind” in the context of an aptamer refers to aprocess of establishing a non-covalent, sequence-specific interactionbetween nucleic acid strands in the aptamer molecule and either proteinor whole cell of the target to form conjugate.

As used herein, the term “probe” refers to a surface-immobilizedmolecule that can be recognized by a particular target.

As used herein, the term “target” refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species.

As used herein, the term “biological chip,” “chip”, or “bio sensor”refers to a substrate having a surface to which one or more arrays ofprobes is attached.

As used herein, the term “wafer” refers to a substrate having a surfaceto which a plurality of probe arrays is attached. On a wafer, the arraysare physically separated by a distance of at least about a millimeter,so that individual chips can be made by dicing a wafer or otherwisephysically separating the array into units having a probe array.

As used herein, the term “aptamer” refers to nucleic acid moleculeshaving specific binding affinity to molecules through interactions otherthan classic Watson-Crick base pairing. Aptamers, like peptidesgenerated by phage display or monoclonal antibodies, are capable ofspecifically binding to selected targets and modulating the target'sactivity, e.g., through binding aptamers may block their target'sability to function. Created by an in vitro selection process from poolsof random sequence oligonucleotides, aptamers have been generated forover 100 proteins including growth factors, transcription factors,enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDain size (30-45 nucleotides), binds its target with sub-nanomolaraffinity, and discriminates against closely related targets (e.g.,aptamers will typically not bind other proteins from the same genefamily). A series of structural studies have shown that aptamers arecapable of using the same types of binding interactions (e.g., hydrogenbonding, electrostatic complementarity, hydrophobic contacts, stericexclusion) that drive affinity and specificity in antibody-antigencomplexes.

As used herein, the term “hairpin structure” refers to anoligonucleotide that contains a double-stranded stem segment and asingle-stranded loop segment wherein the two polynucleotide or nucleicacid strands that form the double-stranded stem segment is linked andseparated by the single polynucleotide or nucleic acid strand that formsthe loop segment. The “hairpin structure” can also further comprise 3′and/or 5′ single-stranded region(s) extending from the double-strandedstem segment.

As used herein, the phrase “two perfectly complementary nucleotidesequences” refers to a nucleic acid duplex wherein the two nucleotidestrands match according to the Watson-Crick base pair principle, i.e.,A-T and C-G pairs in DNA:DNA duplex and A-U and C-G pairs in DNA:RNA orRNA:RNA duplex, and there is no deletion or addition in each of the twostrands.

As used herein, the term “nano-textured” to surfaces having at least onedimension on the nanoscale, i.e., only the thickness of the surface ofan object is between 0.1 and 100 nm.

Accordingly, in one embodiment, the invention is directed to devices,comprising: a thermally responsive, electrically insulating substrate;at least one heating element; and a first detecting unit, comprising: afirst electrode and a second electrode separated by a nanogap; and aplurality of first aptamer probes attached to said substrate in saidnanogap. In certain embodiments, the first aptamer probes aredouble-stranded nucleic acids and have the same nucleic acid sequence.In certain other embodiments, the first aptamer probes comprise: a firstnucleic acid portion in a hairpin loop formation; wherein said firstnucleic acid portion comprise a spacer, a loop, and a stem region, saidstem region being double-stranded; and a second nucleic acid portion ina linear formation; wherein said second nucleic acid portion is singlestranded and is attached to said substrate; and wherein said secondnucleic acid portion is complementary to at least a portion of saidspacer in said first nucleic acid portion; and wherein each of saidfirst aptamer probes has the same nucleic acid sequence.

In yet another embodiment, the invention is directed to systems,comprising: a device described herein; and an electrical reading devicefor interrogating said device; wherein said electrical reading device isoptionally portable.

In one embodiment, the invention is directed to methods for detectinghybridization of a target, comprising: providing a device, comprising: athermally responsive, electrically insulating substrate; at least oneheating element; and a first detecting unit, comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof first aptamer probes attached to said substrate in said nanogap;providing a solution comprising said target under hybridizingconditions; wherein said target is a protein (such as biomarkers,environmental samples, bioterrorism agent like virus and bacteria, andcontaminants) or a cell (such as a tumor cell, especially a circulatingtumor cell (CTC)); and wherein said target hybridizes at least some ofsaid first aptamer probes; applying a voltage drop across saidelectrodes; and measuring a change in conductivity, resistivity,capacitance, or impedance across said electrodes at known locations todetermine perfect complementarity of said target to said first aptamerprobes.

In another aspect, methods of detecting tumor cells are describedherein. In some embodiments, a method of detecting tumor cells comprisescontacting a plurality of cells with a substrate of a device describedherein. In some cases, the device comprises a substrate surface and aplurality of first aptamer probes attached to the substrate surface.Moreover, a method of detecting tumor cells described herein can furthercomprise contacting a plurality of cells with the plurality of firstaptamer probes attached to the substrate surface; adhering one or moreof the plurality of cells to the substrate surface; and measuring anon-uniformity parameter, a Hausdorff distance, a change in the numberof pseudopods, and/or a shape of at least one adhered cell. Moreover, insome instances, such a method further comprises using the non-uniformityparameter, Hausdorff distance, change in the number of pseudopods,and/or shape of the adhered cell to identify the adhered cell as a tumorcell or a non-tumor cell.

Devices

In one embodiment, the invention is directed to devices, comprising: athermally responsive, electrically insulating substrate; at least oneheating element; and a first detecting unit, comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof first aptamer probes attached to said substrate in said nanogap. Incertain embodiments, the first aptamer probes are double-strandednucleic acids and have the same nucleic acid sequence. In certain otherembodiments, the first aptamer probes comprise: a first nucleic acidportion in a hairpin loop formation; wherein said first nucleic acidportion comprise a spacer, a loop, and a stem region, said stem regionbeing double-stranded; and a second nucleic acid portion in a linearformation; wherein said second nucleic acid portion is single strandedand is attached to said substrate; and wherein said second nucleic acidportion is complementary to at least a portion of said spacer in saidfirst nucleic acid portion; and wherein each of said first aptamerprobes has the same nucleic acid sequence.

In certain embodiments, including those involving arrays, the devicefurther comprises: a plurality of second detecting units, comprising: afirst electrode and a second electrode separated by a nanogap; and aplurality of second aptamer probes attached to said substrate in saidnanogap. In certain embodiments where the target is a protein, thesecond aptamer probes are double-stranded nucleic acids and have thesame nucleic acid sequence; wherein said second aptamer probes are thesame or different from said first aptamer probes in said first detectingunit; and wherein said second aptamer probes are the same or differentfrom other second aptamer probes in said plurality of second detectingunits. In certain embodiments where the target is a cell, each of saidsecond aptamer probes comprises: a first nucleic acid portion in ahairpin loop formation; wherein said first nucleic acid portion comprisea spacer, a loop, and a stem region, said stem region beingdouble-stranded; and a second nucleic acid portion in a linearformation; wherein said second nucleic acid portion is single strandedand is attached to said substrate; and wherein said second nucleic acidportion is complementary to at least a portion of said spacer in saidfirst nucleic acid portion; wherein said second aptamer probes are thesame or different from said first aptamer probes in said first detectingunit; and wherein said second aptamer probes are the same or differentfrom other second aptamer probes in said plurality of second detectingunits.

In certain embodiments, the device further comprises: a plurality ofmicrofluidic channels; and an optional cover.

In certain embodiments of the device, the first detecting unit islocated on the surface of said substrate.

In certain embodiments of the device, said at least one heating elementis located in a first layer; and said first electrode and said secondelectrodes are located in a second layer.

The device may be fabricated as a single layer or, preferably, asmultiple layer, using standard and advanced silicon fabricationtechniques. In preferred embodiments, there are two functional layers.The first layer has heating elements, preferably embedded heatingnano-plates. The second layer has electrical nano-electrodes.

Standard complementary metal-oxide-semiconductor (CMOS) processes may beused to create an array of detecting units on a single wafer formultiple nucleic acid detection with printed circuit board (PCB) dataacquisition and analysis capability.

The design of the chip gives an array of detecting units in whichhundreds (n²) of interaction sites may be addressed using a few (2n)probing pads. The probing pads in turn are addressed and controlledusing sensitive electronics and software in the manner that pixels in athin film transistor (TFT) television. This provides an integratedsystem with on-chip circuitry for data gathering, storage, and analysis.Suitable techniques for addressing the interaction sites at theelectrodes in the array are described in the following references, whichare incorporated herein by reference in their entirety: A. Hassibi andT. H. Lee, IEEE Sensors Journal, 6(6), 1380-1388, (2006); W. F. Aerts,S. Verlaak, and P. Heremans, IEEE Transactions on Electron Devices,49(12), 2124-2130, (2002); and A. Hassibi, “Integrated Microarrays”Section in Electrical Engineering. 2005, Stanford University: Palo Alto,Calif. p. 141.

Substrate

The devices of the invention include a thermally responsive,electrically insulating substrate. The substrate is preferably flat butmay take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whichthe probes are located. The substrate and its surface preferably form arigid support on which the probes can be attached. For instance, thesubstrate may be any thermally responsive, electrically insulatingmaterials. Suitable substrates include, but are not limited to,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,polydimethylsiloxane (PDMS), or any one of a wide variety of gels orpolymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, polypropylene, or combinations thereof. Thesubstrate may be deposited by chemical vapor deposition. Other substratematerials and deposition methods will be readily apparent to those ofskill in the art upon review of this disclosure. In a preferredembodiment, the substrate is flat glass or silica with a silicon dioxidelayer grown on the surface to provide electrical insulation.

In certain embodiments, the substrate is nano-textured.Three-dimensionally nano-texturing of the substrate provides a higherprobability of capturing and isolating proteins and/or cells, such astumor cells, from solution, thereby aiding in the development ofcytological tools for circulating tumor cells (CTC) detection, forexample. In certain preferred embodiments, the substrate isnano-textured PDMS.

The aptamer probes useful in the devices, systems, and methods of theinvention are attached to the surface of the chip. In one embodiment,the chip is first cleaned, for example, using oxygen plasma at 200 W inAr+O₂. This treatment also makes the surface hydrophilic. Surfacefunctionalization of the chips may be done in a nitrogen glove box withcontrolled temperature, as described in S. M. Iqbal, D. Akin, and R.Bashir, Nature Nanotechnology, 2(4), 243-248 (2007), incorporated hereinby reference. For example, on a clean chip, a self-assembled monolayer(SAM) of 3-aminopropyltrimethoxysilane (APTMS) (5% solution in 95%ethanol) is reacted with the hydroxyls on the surface to provide silanefunctionality to the surface of the chip. Next, thesilane-functionalized surface is reacted with p-phenylenediisothiocyanate (PDITC)(dissolved in dimethylformamide containing 10%pyridine) as a bifunctional linker. Finally, the chip is immersed in asolution containing the hairpin loop oligonucleotide probes. Onceattached, the probe molecule may be heat-cycled in buffer, such asTrisEDTA (TE) buffer, multiple times to ensure that all molecules formthe requisite hairpin loop structures. To deactivate the unreactedsurface-bound isothiocyanate groups, the chips may be immersed in ablocking solution, such as 50 mM 6-amino-1-hexanol and 150 nM DPEA indimethylformamide (DMF) for 2 hours after which the substrate issequentially washed with, for example, DMF, acetone, deionized water,and dried in a stream of nitrogen.

Heating Elements

In certain embodiments, the heating element is embedded in saidsubstrate. FIGS. 4A and 4B show one embodiment where the heatingelements are embedded in the substrate.

The heating elements useful in the devices, systems, and methods of theinvention may comprise any material suitable for preparing electrodes,including but not limited to, gold, silver, titanium, copper, andcombinations thereof.

In certain embodiments, the devices of the invention have an array ofheating elements. The heating elements are metal nanoplate heaterelements 2 a, 2 b embedded on silicon dioxide chip 1, as shown in FIG.1A and FIG. 1B. The heating elements may be fabricated using standardlithography and can provide precise control of temperature fields, asdescribed in A. Jain, K. D. Ness, H. A. Fishman, and K. E. Goodson,Microtechnology in Medicine and Biology, 2005, 3^(rd) IEEE/EMBS SpecialTopic Conference on 2005: 398-399, incorporated herein by reference.FIG. 2 shows typical temperature gradients from individual nanoplates 2a, 2 b. The individual heating elements may be addressed in groups toenable temperature cycling for various operations of the device,including, but not limited to, focusing of oligonucleotide targetstoward the nanogap between the sets of electrodes 5 a, 5 b, melting ofdouble stranded oligonucleotide targets (without melting and denaturingthe hairpin loop oligonucleotide probes causing loss of sensitivityagainst mismatches), and recycling of the oligonucleotide probes.

In operation as shown in FIGS. 3A and 3B, a solution containing at leastone buffer and the double-stranded oligonucleotide (DNA, cDNA, or RNAfragment) is flowed into the denaturation chambers (not shown) from themicrofluidic channel (not shown) at a temperature gradient at least ashigh as the melting temperature (T_(m)) of the double-strandedoligonucleotide target before being carried over the nanogap of theelectrodes. The black arrows indicate the flow of the solution.

The embedded heating elements serve multiple purposes, including:on-chip/device denaturing of the double-stranded oligonucleotide targetmolecules, removal of secondary structures of cDNA, competitive capturewith the surface-bound oligonucleotide probe; recycling capability ofthe device for the next batch of target solution by denaturing theprobe-target duplex; and temperature gradient focusing to concentratethe solution containing the oligonucleotide target at the nanogap of theelectrodes.

The heating elements may be covered in a thermally-responsive butelectrically-insulating material layer, such as silicon dioxide,deposited, for example, by chemical vapor deposition (CVD).

Electrodes and Nanogap

In certain embodiments, the first and second electrodes comprise a metalselected from the group consisting of gold, silver, titanium, copper, ora combination thereof.

Nanoimprint lithography may be used to fabricate the electrodes with thenanogaps. The electrodes may formed into an array where each nanogap isindividually addressed with metal lines (bus-bars), preferably runningat right angles. Each mutually-insulated intersection of the addressinglines contacts one nanogap electrode pair that serves as the binding andsensing site of the probe-target hybridization. The bus lines may befabricated in two layers with electrical isolation between the twolayers achieved by chemical vapor deposition (CVD) of silicon nitride.FIG. 5A is a top view of one embodiment showing two bus lines 9 a, 9 bused to address the individual set of electrodes 5 a, 5 b with a nanogap6 fabricated with nanoimprint lithography. Arrows indicate the extensionof similar structures to cover the whole chip area. In certainembodiments, microfluidic channels run exactly on top of the nanogapelectrodes, thus reducing any cross-talk between probe and targetmolecules of successive rows.

FIG. 5B is a blown up schematic (not to scale) of FIG. 5A showing theindividual set of electrodes 5 a, 5 b with a nanogap 6, one electrodeaddressing/measuring bus 9 a covered with silicon nitride and the otherelectrode addressing/measuring bus 9 b. Oligonucleotide probe molecules(not shown) are attached to the chip in the nanogap 6 between theelectrodes 5 a, 5 b. This three-dimensional structure is adjacent to andaligned with embedded nanoplate heating elements discussed previously.The structure is covered with microfluidic channels. The electricalisolation may be achieved by sequential and automated measurement ofeach pair of electrodes. In preferred embodiments, the use of chemicalvapor deposition (CVD) silicon dioxide results in a rough surface whichfacilitates the covalent attachment of the oligonucleotide probes to thesurface of the substrate, thereby permits an increased density of probesin the nanogap.

In certain embodiments, nanopatterns may be made on an oxidized siliconwafer using e-beam lithography (EBL). The EBL patterns may be used tofabricate the stamp for the nanoelectrode fabrication. The EBL patternsmay be used to remove silicon dioxide and then silicon from thenon-patterned areas using deep reactive ion etching (DRIE). Silicondioxide acts as a hard mask during the process, resulting is a highaspect ratio nano-scale linear island features in silicon having thesame dimensions as required the nanogap electrodes. The wafer may act asa stamping mask for NIL. In NIL, a polymer layer is spun on the waferand a stamping wafer is compressed on the polymer to transfer thepattern. One stamp can be used multiple times and one stamping takes afew minutes to transfer the nano-scale patterns in the polymer. Standardlift-off process may be carried out to create the metal lines at thenanoscale from these stamp-defined patterns. In certain embodiments ofthe lift-off process, metal stays only in the NIL transferrednanoelectrode structure and the remainder of the metal lifts off in anultra-sonicator assisted solvent soak. The first layer of addressingelectrodes/bus may then be using standard optical lithography aligned tothe nano-scale metal lines. The second layer of metal lines/bus may bedeposited after CVD deposition of silicon nitride and reactive ion etchopening of small micron sized windows in the silicon nitride above thenanogap electrodes.

The use of three-dimensional interaction volume and interfacing spaceaddresses the challenges of selectivity, high-yield fabrication, andsensitivity. Such three-dimensionality provides a means of not onlyimproving selectivity by filtering unwanted species, but also allowsreduced signal-to-noise ratios relative to what is attainable on planarsubstrates. The nanoelectrodes employed in the devices of the inventionenable the majority of the electric field to interact with thebiomolecules, resulting in highly sensitive detection.

Different methods may be used to fabricate the nanogap between a set ofelectrodes. A break junction may be prepared from an already fabricatedelectrode by, for example, a mechanically controlled break junctionprocess or an electromigration break junction process. In certainpreferred embodiments, wherein said nanogap is formed by focused ionbeam scratching followed by electromigration. FIG. 7 shows an scanningelectron micrograph of a set of electrodes of gold with a nanogap.

In certain embodiments, the nanogap is about 10 nm to about 500 nm.

A break junction is the discontinuity or a nanoscale gap in a seeminglycontinuous structure. The most common visualization of a break junctionis a gap formed in a thin metal strip by various methods. The nanoscalegap in the metal strip is the area of interest where the molecule isplaced to make electrical contacts.

A mechanically-controlled break junction process is a process where anarrow bridge of metal is suspended above a flexible substrate. Bybending the substrate, the metal bridge can be broken, and the distancebetween the ends can be controllably adjusted, with increments of muchless than a pico meter.

To create a break junction using electromigration, an external electricfield applied to a circuit causes large current density in the wiresthat connect the components. The electrons in a metal move under theinfluence of the large current density and if there is a charged defectin the metal, the momentum transfers from the conduction electrons tosuch a defect. As the momentum exchange becomes larger, a force is builtup causing the mass movement of the atoms away from the defect causingbreakdown of the metal at that point. In break junction, the breakusually occurs in the constricted part of the metal in a controllableand self-limiting fashion. Normally, the breaking process consistentlyproduces two metallic electrodes at typical separations.

There are several techniques to fabricate break junctions, well known inthe art. Almost all of them follow the conventional process of thin filmdeposition, lithography and etching on oxidized silicon wafers. Suitabletechniques include those disclosed in the following references, whichare incorporated herein by reference in their entirety: Park, H., etal., Applied Physics Letters, 75: 301-303 (1999); Zhou, C., et al.,Applied Physics Letters, 67: 1160-1162 (1995); Bezryadin, A. and C.Dekker, Journal of Vacuum Science & Technology B: Microelectronics andNanometer Structures, 15: 793-799 (1997); and Bezryadin, A., C. Dekker,and G. Schmid, Applied Physics Letters, 71: 1273-1275 (1997).

Fabrication of controlled nano-gap electrodes is another efficientmethod of trapping the oligonucleotide probes. This method also followsthe same procedure of integrating single or double strand DNA into acircuit using metal electrodes to perform conductivity measurements. Theseparation between the electrodes must be small in accordance with thelength nucleic acid molecules used.

In certain embodiments, a 7-bit addressing scheme with multiplexercircuits may be used in conjunction with an analog signal for thebiasing of the sensing/measurement block at the nanogap. An externalclock may be used on the mounting printed circuit board to provide themeasurement frequency. The clock enables the input biasing across thesensing/measurement block. The current or conductance in response to thebias is measured at the shared output electrode and may be normalizedand co-related with the preceding and following data gathered from thesame sensing/measurement block.

In certain embodiments, the invention is directed to methods of forminga metallic nano-scale break junction on a chip, comprising: forming onsaid chip a metal line, preferably having a thickness of less than about5 μm and a width less than about 5 μm; bombarding said metal line with afocused-ion beam to form a thinned section in said metal line; andapplying current to said metal line sufficient to cause electromigrationin said thinned section of said metal line. In preferred embodiments,the metal line is formed using photolithography.

Probes

The probes useful in the invention are aptamers. Aptamers of the presentinvention can be obtained by SELEX (Systematic Evolution of Ligands byEXponential enrichment) method, commonly used for obtaining aptamers.First, a template DNA is synthesized that contains an appropriate lengthof random sequence flanked by two arbitrary primer sequences. Thistemplate DNA is amplified by PCR (Polymerase Chain Reaction) to obtain arandomized DNA aptamer pool. Next, the randomized DNA aptamer pool isassociated with a target substance, and then DNAs not bound to thetarget substance are removed, and DNA aptamers bound to the targetsubstance are extracted. The resultant DNA aptamers are amplified by PCRusing the primer sequences, wherein the PCR is performed under thepresence of 5 to 8 mM of Mg2+ for lowering replication accuracy andcausing a mutation to be introduced more easily to obtain a further DNAaptamer pool that contains new DNA aptamers that would not be present inthe DNA aptamer pool before performing the association with the targetsubstance. The new DNA aptamers may have a stronger binding strength,that is, evolved DNA aptamers may be generated. A series of proceduresexplained above is repeated for 5 to 15 rounds with a pool of theevolved DNA aptamers to obtain DNA aptamers being able to specificallybind to the target substance. The resultant DNA aptamer pool after thefinal round is cloned and sequenced as usually performed by thoseskilled in the art. The procedures such as synthesis of template DNA andPCR in the SELEX process and cloning and sequencing are performed bymethods commonly used by those skilled in the art. The aptamers of thepresent invention can be chemically synthesized by methods commonly usedby those skilled in the art based on the determined sequence. Methods ofmaking oligonucleotides of a predetermined sequence are well-known. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed.(Oxford University Press, New York, 1991). Solid-phase synthesis methodsare preferred for both oligoribonucleotides andoligodeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Oligoribonucleotides andoligodeoxyribonucleotides can also be prepared enzymatically.

In certain embodiments where the target is a protein, the first aptamerprobes are double-stranded nucleic acids and have the same nucleic acidsequence.

In certain embodiments where the target is a whole cell, each of saidfirst aptamer probes comprises: a first nucleic acid portion in ahairpin loop formation; wherein said first nucleic acid portion comprisea spacer, a loop, and a stem region, said stem region beingdouble-stranded; and a second nucleic acid portion in a linearformation; wherein said second nucleic acid portion is single strandedand is attached to said substrate; and wherein said second nucleic acidportion is complementary to at least a portion of said spacer in saidfirst nucleic acid portion; and wherein each of said first aptamerprobes has the same nucleic acid sequence. In certain embodiments, eachof said first aptamer probes comprises: a spacer having about onenucleotide base to about 20 nucleotide bases; a nucleotide loop havingabout 3 nucleotide bases to about 100 nucleotide bases; and a base pairstem having about 3 nucleotide base pairs to about 20 nucleotide basepairs. In certain embodiments, each of said first aptamer probescomprises: a spacer having about 3 nucleotide bases; a nucleotide loophaving about 12 nucleotide bases; and a base pair stem having about 6nucleotide base pairs.

In certain embodiments, the aptamer probes are covalently attached tosaid substrate.

In one embodiment, the aptamer probes are amine-modified to permitcovalent attachment to the substrate. In certain embodiments, there isdirect attachment of the oligonucleotide probe through a silane, suchas, for example, (3-glycidyloxypropyl)trimethoxysilane). In otherembodiments, there is attachment through a homo-bifunctional layer, suchas, for example, 1,4-phenylene diisothiocyanate, on top of a silane,such as, for example, 3-aminopropyltrimethoxysilane.

The selection of probes and their organization in an array depends uponthe use to which the biological chip will be put. In one embodiment, thechips are used to sequence or re-sequence nucleic acid molecules, orcompare their sequence to a reference molecule. Re-sequencing nucleicacid molecules involves determining whether a particular molecule hasany deviations from the sequence of reference molecule.

In typical diagnostic applications, a solution containing one or moretargets to be identified (i.e., samples from patients) contacts theprobe array. The targets will bind or hybridize with complementary probesequences. Accordingly, the probes will be selected to have sequencesdirected to (i.e., having at least some complementarity with) the targetsequences to be detected, e.g., human or pathogen sequences.Accordingly, locations at which targets hybridize with complimentaryprobes can be identified by locating the electrical current in anelectrode set. Based on the locations of the electrodes wherehybridization occurs, information regarding the target sequences can beextracted. The existence of a mutation may be determined by comparingthe target sequence with the wild type.

Individual probe sequence may be designed to detect known singlemutations. However, the invention is not limited to methods of detectingknown single mutations, but may be used to identify the sequence of anydesired target.

The aptamer probes may be immobilized on the substrate between theelectrodes using the surface chemistry described by the techniquesdescribed in M. Manning, S. Harvey, P. Galvin, and G. Redmond, MaterialsScience and Engineering, C 23, 347 (2003), incorporated herein byreference.

In certain embodiments, the aptamer probes form a self-assembledmonolayer between the electrodes.

The aptamer probes may be directed to and located in the gaps between aparticular set(s) of electrodes by electrostatic trapping, i.e.,energizing a particular set(s) of electrodes, preferably usingalternating current, to direct the probes (which are charged) to thegaps. Different oligonucleotide probes, referred herein as “secondaptamer probes,” may be localized at their respective electrodes byproviding the second aptamer probes and sequentially energizing thedesired set(s) of electrodes to direct and localize the secondoligonucleotide probes in the appropriate nanogap(s). This procedure maybe repeated until all of the different aptamer probes are directed toand located in the nanogap(s) between their desired set(s) ofelectrodes.

Micro Fluidic Channels

Assays on biological arrays generally include contacting a probe arraywith a sample under the selected reaction conditions, optionally washingthe well to remove unreacted molecules, and analyzing the biologicalarray for evidence of reaction between target molecules and the probemolecules. These steps involve handling fluids. Microfluidic channels 15may be used to deliver the liquids to the test sites, such as the oneshown in FIG. 6 made from any suitable solid material, such aspolydimethylsiloxane. The methods of this invention automate these stepsso as to allow multiple assays to be performed concurrently.Accordingly, this invention employs automated fluid handling systems forconcurrently performing the assay steps in each of the test wells. Fluidhandling allows uniform treatment of samples in the test sites.Microtiter robotic and fluid-handling devices are availablecommercially, for example, from Tecan AG.

The device may be introduced into a holder in the fluid-handling device.This robotic device may be programmed to set appropriate reactionconditions, such as temperature, add samples to the device, incubate thetest samples for an appropriate time, remove unreacted samples, wash thewells, add substrates as appropriate and perform detection assays. Theparticulars of the reaction conditions depend upon the purpose of theassay. For example, in a sequencing assay involving aptamerhybridization, standard hybridization conditions are chosen. However,the assay may involve testing whether a sample contains target moleculesthat react to a probe under a specified set of reaction conditions. Inthis case, the reaction conditions are chosen accordingly.

Systems

In other aspects, the invention is directed to systems, comprising: adevice described herein; and an electrical reading device forinterrogating said device described herein. In certain preferredembodiments, said electrical reading device is portable.

Preferably, the optional linker further comprises a hydrocarbon moietyattached to the cyclic disulfide. Suitable hydrocarbons are availablecommercially, and are attached to the cyclic disulfides. Preferably thehydrocarbon moiety is a steroid residue. Oligonucleotide-nanoparticleconjugates prepared using linkers comprising a steroid residue attachedto a cyclic disulfide are stable to thiols (e.g., dithiothreitol used inpolymerase chain reaction (PCR) solutions) as compared to conjugatesprepared using alkanethiols or acyclic disulfides as the linker. Thisstability is likely due to the fact that each oligonucleotide isanchored to a nanoparticle through two sulfur atoms, rather than asingle sulfur atom. In particular, it is thought that two adjacentsulfur atoms of a cyclic disulfide would have a chelation effect whichwould be advantageous in stabilizing the oligonucleotide-nanoparticleconjugates. The large hydrophobic steroid residues of the linkerscontribute to the stability of the conjugates by screening thenanoparticles from the approach of water-soluble molecules to thesurfaces of the nanoparticles.

In view of the foregoing, the two sulfur atoms of the cyclic disulfideshould preferably be close enough together so that both of the sulfuratoms can attach simultaneously to the nanoparticle. Most preferably,the two sulfur atoms are adjacent each other. Also, the hydrocarbonmoiety should be large so as to present a large hydrophobic surfacescreening the surfaces of the nanoparticles.

Electrical Reader

Suitable electrical reading devices include any device for low powerprinted circuit board electronics capable of measuring eithersequentially or in parallel a small change in conductivity, resistivity,capacitance, or impedance in a picoampere range. The Agilent 4155Csemiconductor parameter analyzer and the Agilent 4156C semiconductorparameter analyzer are examples of suitable devices.

Methods

The invention provides a nanotechnology-based low-power, rapid,inexpensive, recyclable, and sensitive electrical detection device,system, and method of sub-femtomolar concentrations of proteins andcells, with no external sample preparation or labeling or other chemicalmodification of the sample. The biosensors of the invention may be usedin wide variety of applications requiring sensitive target detection,including, but not limited to, forensics, early disease detection,disease progression monitoring (such as in response to therapy and/ormedicinal agents), legal matters (such as paternity and criminalproceedings), defensive biohazard detection, and immigration issues(such as establishing blood relationships). The biosensors of theinvention are useful in further enabling “personalized medicine,” wheredrugs are designed according to each individual's genetic make-up.

In another aspect, the invention is directed to methods for detectinghybridization of a target, comprising: providing a device, comprising: athermally responsive, electrically insulating substrate; at least oneheating element; and a first detecting unit, comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof first aptamer probes attached to said substrate in said nanogap;providing a solution comprising said target under hybridizingconditions; wherein said target is a protein or a cell; and wherein saidtarget hybridizes at least some of said first aptamer probes; applying avoltage drop across said electrodes; and measuring a change inconductivity, resistivity, capacitance, or impedance across saidelectrodes at known locations to determine perfect complementarity ofsaid target to said first aptamer probes.

In certain embodiments, the methods further comprise: washing to removeunhybridized components from said detecting unit.

In certain embodiments, the methods further comprise: heating saiddevice to remove said hybridized targets and said hybridizednanoparticle reporter conjugates from said probe to permit recycling ofsaid detecting unit.

In certain embodiments, the methods further comprise: heating a solutioncomprising double stranded oligonucleotide target to form said solutioncomprising single-stranded oligonucleotide target.

In certain embodiments, the methods further comprise: forming atemperature gradient to focus said single stranded oligonucleotidetarget at said detecting unit.

In certain embodiments, the methods further comprise: reversing thepolarity of said voltage drop to remove unbound components ornonspecifically bound components from said detecting unit.

The methods of the invention may be used to quantify the level ofoligonucleotides or polypeptides. For example, the change in conductance(or other electrical characteristic) between nanogaps is direct functionof the number of specific binding events between nanogaps. The number ofnanoparticles is a direct function of the number of specific bindingevents. Thus, the change of conductivity (or other electricalcharacteristic) can be directly correlated to the quantity of specificbinding between the proteins or cells present in the sample and theaptamer probes.

In certain embodiments, the methods further comprise: providing, inaddition to said first detecting unit, a plurality of additionaldetecting units, each additional detecting unit comprising: a firstelectrode and a second electrode separated by a nanogap; and a pluralityof second oligonucleotide probes attached to said substrate in saidnanogap; wherein said second oligonucleotide probes are in a hairpinloop formation and have the same nucleic acid sequence; and wherein saidsecond oligonucleotide probes comprise an optional spacer, a loop, and astem region, said stem region being double-stranded; wherein said secondoligonucleotide probes are the same or different from said firstoligonucleotides in said first detecting unit; and wherein said secondoligonucleotide probes are the same or different from other secondoligonucleotide probes in said plurality of second detecting units;providing a plurality of at least one second nanoparticle reporterconjugates under hybridizing conditions; wherein said secondnanoparticle reporter conjugates comprise at least one nanoparticle andan oligonucleotide complementary to at least a portion of said stem ofsaid second oligonucleotide probes; wherein said second nanoparticlereporter conjugates are the same or different from said firstnanoparticle reporter conjugates; wherein said second nanoparticlereporter conjugates are the same or different from said other secondnanoparticle reporter conjugates; wherein said measuring step is carriedout in parallel or sequentially for said first detecting unit and saidplurality of said additional detecting units.

In certain embodiments wherein said target is a protein target, thefirst aptamer probes are single-stranded or double-stranded nucleicacids and have the same nucleic acid sequence. The protein target may beoptionally tagged with a nanoparticle selected from the group consistingof a metal, semiconductor, magnetic colloidal particle, or a combinationthereof.

In certain embodiments wherein said target is a cell target; each ofsaid first aptamer probes comprising: a first nucleic acid portion in ahairpin loop formation; wherein said first nucleic acid portion comprisea spacer, a loop, and a stem region, said stem region beingdouble-stranded; and a second nucleic acid portion in a linearformation; wherein said second nucleic acid portion is single strandedand is attached to said substrate; and wherein said second nucleic acidportion is complementary to at least a portion of said spacer in saidfirst nucleic acid portion; and wherein each of said first aptamerprobes has the same nucleic acid sequence.

In certain embodiments, said voltage drop is applied as direct current.In other embodiments, said voltage drop is applied as alternatingcurrent and the alternating current impedance measured.

In certain embodiments, said measuring step measures an increase inconductivity across said electrodes at known locations to determineperfect specific binding of said protein or cell target to said firstaptamer probes.

In certain embodiments, the method may optionally include the step ofreversibly exchanging an imino proton in each base pair of the firstaptamer probes with a metal ion selected from the group consisting ofgold ion, silver ion, platinum ion, and copper ion. The reversibleexchanging of an imino proton in each base pair may be carried out asdescribed in A. Rakitin, Aich, P., Papadopoulos, C., Kobzar, Yu.,Vedeneev, A. S., Lee, J. S., J. M. Xu, Phys. Rev. Lett., 86(16),3670-3673, (2001), which is incorporated herein by reference.

In certain embodiments, the method may optionally include the step ofvectorially depositing silver on the double stranded nucleic acidsequence of the aptamer probes. In certain embodiments of this method,the vectorially depositing step comprises: ion exchanging silver ions onsaid double stranded aptamer sequence; reducing said silver ions; anddeveloping silver aggregates on said double stranded nucleic acidsequence; as described in E. Braun, Y. Eichen, U. Sivan, and G.Ben-Yoseph, Nature, 391(6669), 775-778, (1998), incorporated herein byreference.

The methods of this invention will find particular use wherever highthrough-put of samples is required. The clinical setting requiresperforming the same test on many patient samples. The automated methodsof this invention lend themselves to these uses when the test is oneappropriately performed on a biological chip. For example, a DNA arraycan determine the particular strain of a pathogenic organism based oncharacteristic DNA sequences of the strain. The advanced techniquesbased on these assays now can be introduced into the clinic. Fluidsamples from several patients are introduced into the test wells of abiological chip plate and the assays are performed concurrently.

In some embodiments, it may be desirable to perform multiple tests onmultiple patient samples concurrently. According to such embodiments,rows (or columns) of the microtiter plate will contain probe arrays fordiagnosis of a particular disease or trait. For example, one row mightcontain probe arrays designed for a particular cancer, while other rowscontain probe arrays for another cancer. Patient samples are thenintroduced into respective columns (or rows) of the microtiter plate.For example, one column may be used to introduce samples from patient“one,” another column for patient “two” etc. Accordingly, multiplediagnostic tests may be performed on multiple patients in parallel. Instill further embodiments, multiple patient samples are introduced intoa single well. In a particular well indicator the presence of a geneticdisease or other characteristic, each patient sample is thenindividually processed to identify which patient exhibits that diseaseor trait. For relatively rarely occurring characteristics, furtherorder-of-magnitude efficiency may be obtained according to thisembodiment.

Particular assays that will find use in automation include thosedesigned specifically to detect or identify particular variants of apathogenic organism, such as HIV. Assays to detect or identify a humanor animal gene are also contemplated. In one embodiment, the assay isthe detection of a human gene variant that indicates existence of orpredisposition to a genetic disease, either from acquired or inheritedmutations in an individual DNA. These include genetic diseases such ascystic fibrosis, diabetes, and muscular dystrophy, as well as diseasessuch as cancer (the P53 gene is relevant to some cancers), as disclosedin U.S. Pat. No. 5,837,832.

Methods of detecting tumor cells and distinguishing tumor cells fromnon-tumor cells are also described herein. Thus, the present disclosureprovides a mechanism for the early detection of cancer. Tumors startshedding cancer cells in the blood stream very early in the disease.These metastatic tumor cells may or may not form secondary tumors atonset of a primary tumor but their presence in blood is notwell-established. These cells are called circulating tumor cells (CTC)or rare cells. Their concentration is too low at early stages and thusit is a challenge to detect them. The shedding of CTCs starts longbefore clinical symptoms of cancer become evident. Thus, there is a needfor simple, cheap, rapid, and non-invasive tests for cancer that can bedone as routine lab-work during annual or biannual physical check-ups.The present disclosure provides a method to check for and/or detect CTCsfrom blood as well as from simple body fluids like urine, saliva, andbladder wash. Moreover, the present disclosure provides a test that canbe done in a simple clinical setup with minimal intervention or need oftechnicians for analysis or sample handling. Thus, in another aspect,improved methods of detecting cancer metathesis are described herein.

In some embodiments, a method of detecting tumor cells comprisesproviding a device described herein, wherein the device comprises asubstrate surface and a plurality of first aptamer probes attached tothe substrate surface. The method further comprises contacting aplurality of cells with the plurality of first aptamer probes attachedto the substrate surface; adhering one or more of the plurality of cellsto the substrate surface; and measuring a non-uniformity parameter, aHausdorff distance, a change in the number of pseudopods, and/or a shapeof at least one adhered cell. Additionally, in some cases, a methoddescribed herein further comprises using the non-uniformity parameter,Hausdorff distance, change in the number of pseudopods, and/or shape ofthe adhered cell to identify the adhered cell as a tumor cell or anon-tumor cell. Moreover, in some embodiments, the plurality of firstaptamer probes can be at least partially replaced by a plurality ofantibodies.

The substrate of a method described herein can comprises any substratenot inconsistent with the objectives of the present disclosure,including a substrate of a device described hereinabove. In some cases,the substrate surface is formed from glass, silicon, or a polymer. Othersurfaces that can be functionalized with aptamers (or antibodies) mayalso be used. Further, in some instances, the plurality of first aptamerprobes forms a uniform or substantially uniform coating on the substratesurface. A “uniform” coating can cover the entirety of the substratesurface. A “substantially uniform” coating can cover at least about 70%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 95% of the substrate surface. For example,in some embodiments, at least about 80% of the substrate surface iscoated by the plurality of first aptamer probes.

Moreover, the aptamer probes can comprise any aptamer probes describedhereinabove. In some cases, for instance, the plurality of first aptamerprobes comprises an anti-EGFR aptamer.

In addition, the plurality of cells analyzed by a method describedherein can comprise tumor cells, non-tumor cells, or a mixture of tumorcells and non-tumor cells. In some cases, tumor cells comprise CTCs. Insome embodiments, tumor cells comprise breast cancer cells, cervicalcancer cells, lung cancer cells, bladder cancer cells, ovarian cancercells, esophageal cancer cells, head and/or neck cancer cells, kidneycancer cells, glioma cells, bladder cancer cells, pancreatic cancercells, colon cancer cells, or a combination thereof.

Further, in some cases, contacting a plurality of cells with theplurality of first aptamer probes (or antibodies) comprises contacting abodily fluid with the plurality of first aptamer probes (or antibodies).Any body fluid not inconsistent with the objectives of the presentdisclosure may be used. In some cases, for instance, the bodily fluidcomprises blood, saliva, urine, or bladder wash.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present invention andspecific examples provided herein without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncover the modifications and variations of this invention that comewithin the scope of any claims and their equivalents.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1

The “break junction” provides a way to interrogate electrical transportproperties of molecules. Selective probe molecules are immobilizedbetween the contact structures with nanometer sized separation. Breakjunction fabrication was used, including focused ion beam (FIB)“scratching” followed by electromigration, producing elegant, rapid andcontrolled high-yield nano-manufacturing of break junctions at exactlocations with very narrow distribution of the gaps (betweenelectrodes). FIB was used to introduce defects in a lithographicallydefined metal line by scratching the line surface at specific location.The scratch results in high resistance at that particular scratched partand induced electromigration results in a break at that exact location.Gaps ranging between 100-200 nm have been reproducibly prepared. Thebreak junctions were then functionalized with RNA aptamer molecules andare used to detect an important cancer biomarker Epidermal Growth FactorReceptor (EGFR). EGFR over-expression is known in several types ofcancers like breast cancer, lung cancer, cervical cancer, bladdercancer, esophageal cancer, and ovarian cancer. The identification ofEGFR as a common element of several cancer types and it being the mostcommon oncogene emphasizes the implication of detecting EGFR at anearlier stage for early diagnosis and better treatment of cancerpatients.

A. Method

The process comprises two-step photolithography for 3 micron lines andthe probing pads (FIG. 8). FIB “scratching” was used to remove a thinlayer of metal from the lines. The novelty lies in using opticallithography to define the lines, and instead of low-throughput e-beamwriting of the full electrode features, FIB writes a 50 nm wide scratch,reducing time to fabricate and increasing throughput. The FIB processdepends on applied acceleration voltage, milling current and scratchingtime. At higher milling current and scratching time, the entire goldlayer got removed and also induced defects in SiO₂ layer beneath,rendering device un-usable. Secondly the higher milling current producedlarger gaps (FIG. 9). FIB process was optimized for narrow breakjunctions. FIG. 10 shows micrograph of a representative break junctionafter electromigration. The electromigration results from theapplication of an external voltage which causes a large current densityin the metal lines. When the drifting electrons encounter FIB induceddefects, the momentum of the electrons is transferred to the defectswhich results in the build-up of a force that causes atoms to move awayfrom the defect culminating in the break junction formation. Weachieved >60% yield, in comparison to reported yield of <20% byelectromigration-only.

The single stranded DNA (ssDNA) were immobilized on the chemicallymodified silicon dioxide (SiO₂) surface underneath the patterned breakjunctions. The selective RNA aptamers were hybridized with the surfacebound ssDNA. After that, the chips were incubated in EGFR buffersolution to capture the protein.

B. Analysis

Current-Voltage (I-V) measurements of break junctions were done beforeand after FIB scratch, and after electromigration using Agilent 4155CSemiconductor Parameter Analyzer. Following the scratch, a rampingvoltage was applied and a sudden drop was seen in the current,representing the complete break, and noticed from complete loss ofconductivity (FIG. 11). Once EGFR was selectively captured between theelectrodes, increase in the conductivity through the devices was noticeddue to the conducting behavior of proteins that bridge the nanogapbetween the metal electrodes (FIG. 12). The selectivity of the anti-EGFRRNA aptamer to the cancer biomarker was revealed using control chipswith no aptamers. The Conductance-Voltage (G-V) data from −1 to 1 Vacross the electrodes did not show significant change in electricalconductance after EGFR incubation (FIG. 13).

The aptamers are essentially DNA or RNA molecules that are iterativelyselected for their specific binding with nucleic acids, proteins, andsmall organic compounds. Apart from their natural exclusion againstbiofouling, aptamers can be chemically synthesized, and can belabeled/loaded with various reporters/payload. The aptamers are alsostable at various salinity, temperature and surface functionalizationsunlike antibody functionalization of devices. Anti-EGFR aptamers havebeen previously used for the isolation and enrichment of cancer cells.In the current context, the electrical detection of EGFR, selectivelycaptured with surface-bound anti-EGFR aptamer, provides another earlydiagnosis modality against cancer. EGFR is a large, monomeric, modularglycoprotein with its dimensions on the order of tens of angstroms (FIG.14). It is expected that the protein structure would expand when itbinds to the probe aptamer via its extracellular ligand binding domain.This provides a low demand on the break junction size stringency;proteins (biomarkers or others) even with the break junctions that arelarger than just a few nanometers may be detected.

In other words, protein detection does not require sub-10 nm dimensionsof break junctions as made with e-beam lithography. Thus, breakjunctions have come a long way from simple electronic signature studiesof atoms/molecules to the detection of highly complex molecules likemedically-relevant proteins.

Example 2

A. Materials

The chemicals used were 3′-aminopropyltrimethoxy-silane (APTMS);1,4-phenylene diisothiocyanate (PDITC); N,N-dimethylformamide (DMF);1,2-dichloroethane; N,N-diisopropylethylamine (DPEA); 6-amino-1-hexanol;and methanol. Autoclaved de-ionized water (DIW) was used to make thebuffer solutions. The chemicals were purchased from Sigma-Aldrich (SaintLouis, Mo.). The 3′-amino modified DNA strands were purchased from AlphaDNA (Montreal, Quebec). The DNA binding domain from the Bombyx moriretrotransposon protein R2Bm was prepared. The zinc finger and myb motifof the R2Bm protein binds to a specific dsDNA sequence(5′-CTTAAGGTAGCAAATGCCTCGTC-3′) (SEQ ID NO:6) within the gene coding forthe large ribosomal subunit.

B. Method

The reported work consists of three major parts that were carried out inparallel: a) Fabrication of the CMOS chip; b) SiO₂ surface preparation,modification and attachment of dsDNA on the Chip; c) Binding betweenprotein and DNA and optical/electrical detection.

a) Fabrication of the CMOS Chip:

Chips were fabricated in two steps of lithography. On the first layersTi/Au (Thickness 50 Å/150 Å) metal pads 500 nm apart were made usinge-beam lithography. Metal lift-off resulted in well-defined structures.In the second step, optical lithography was used to fabricate probingpads to contact the thin film electrodes. The chips were partiallyfabricated at Birck Nanotechnology Center (Purdue University),Nanotechnology Core Facility (University of Illinois at Chicago) andNanotechnology Research and Teaching Facility (University of Texas atArlington). The e-beam lithography was done by xlith (Ulm, Germany).

b) Surface Modification and Attachment of dsDNA:

The silicon chip was cleaned using UV Ozone plasma system. This alsoresulted in hydrophilic SiO₂ surface. The attachment chemistry wasperformed in a nitrogen glovebox in a controlled environment. Briefly,the chips were silanized in a 3% APTMS solution (made with 19:1methanol-DIW solution) for over 12 hours. The chips were then cured at110° C. for 15 minutes. These were then washed with methanol, DIW anddried with nitrogen gas. The chips were then immediately immersed in aDMF solution containing 10% pyridine and 1 mM PDITC overnight. Afterthis, the chips were sequentially washed with DMF and 1,2-dichloroethaneand dried under nitrogen gas. The dsDNA sequence solution was preparedat a concentration of 1 pmole/μl and chips were immersed in itimmediately. The chips were incubated at 37° C. overnight in order tofacilitate the covalent attachment of the 3′-amino modified dsDNA withthe PDITC cross linker molecules. The chips were again washed with DIW,methanol and dried under nitrogen. The unbound reactive groups fromPDITC were deactivated by immersing the chips in a solution of 50 mM6-amino-1-hexanol and 150 mM DPEA in DMF for 24 hours. The chips werethen washed with DMF, acetone, DIW and dried with nitrogen gas.

In order to confirm the surface modification, Energy-dispersive X-rayspectroscopy (EDAX), contact angle and ellipsometry measurements werecarried out at every step. The presence of dsDNA immobilized on thesilicon surface was confirmed by fluorescence measurements of AcridineOrange stain at 525 nm wavelength using, using Zeiss ConfocalMicroscope.

c) Binding Between Protein and DNA:

The dsDNA used in these experiments was a 23 base-pair (bp) fragment ofthe ribosome gene that corresponds to the binding site of the R2Bmderived polypeptide. In order to confirm that the purified R2Bmpolypeptide is capable of binding to the short dsDNA, an electrophoreticmobility shift assay (EMSA) was run.

Chips with covalently attached dsDNA were then incubated with 2.8fmole/μl of R2Bm polypeptide for 30 minutes in binding buffer (50 mMTris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl₂). The presence of the proteinon the chip was initially confirmed by optical detection of fluorescentSypro Ruby Protein Blot stain at 488 nm wavelength. The fluorescenceintensity analysis was done with ImageJ software. The presence ofprotein bound to the dsDNA was also detected electrically by directcurrent electrical measurements.

C. Results

The EDAX analysis was used to identify the elemental composition of thesilicon surface as the different modifications were added. The data inTABLE I show the elemental increase in Carbon and Nitrogen after dsDNAimmobilization. Control chips without dsDNA showed no change in carbonand nitrogen.

TABLE I EDAX Analysis - Weight % of Significant Elements on Chips withand without Modifications C N O Clean Chip 0.2 8.2 320.4 PDITC 7.3 9.1329.2 DNA 10.7 25.8 391.4

The contact angle measurements showed the silicon surface becominghydrophilic after plasma treatment and later less hydrophilic whenfunctionalized with APTMS and PDITC. This showed that the surface ofsilicon chip was hydroxyl (—OH) rich after plasma etching.Functionalization with APTMS/PDITC reduced available —OH groups on thesurface and thus showed reduced hydrophilicity. This also proved that OHbonds were used up in effective covalent attachment of silane.

The ellipsometry measurements gave the thickness of the self-assembledmonolayers (SAM) of silane modification as shown in TABLE II. Thedifference in the two thicknesses is around ˜10 nm. Reaction conditionslike temperature, silane concentration, nature of the aminosilane,solvent type, incubation time and more importantly, the amount ofadsorbed water, all contribute to the reproducibility of the finalstructure of the adsorbed aminosilane layer. Our functionalization setupcarefully maintained these conditions resulting in reproducible results.

TABLE II Ellipsometry Measurements (in nm) Thickness SD Silicon dioxide1203.66 2.18 APTMS 1213.35 7.32

The presence of dsDNA immobilized on the silicon surface was confirmedby Acridine Orange (FIG. 15). Acridine Orange gives a green fluorescencewhen it interacts with dsDNA. The Acridine Orange stain bears a positivecharge and binds electrostatically with the dsDNA. Electrostaticinteractions with non-specific polyanions is avoided by using a very lowconcentration of the stain (0.2% v/v) and by including other cationslike Mg²⁺, Na⁺ in the buffer solution that would compete for the bindingwith the dsDNA. Thus, the Acridine Orange stain fluorescence obtainedcould be taken as a conclusive result of the covalent attachment ofdsDNA on CMOS chip surface.

Prior to functionalizing the chips, the polypeptide binding to the 23 bpdsDNA fragment was confirmed using EMSA—a polyacrylamide gelelectrophoresis based method to detect protein-DNA interactions (FIG.16). Importantly, the peptide binding to the DNA was seen on thefunctionalized chip; FIG. 17 shows the data for the protein stain SyproRuby confirming polypeptide binding to dsDNA on chip. The Sypro Rubystain is a ruthenium based stain that detects the amino acids lysine,Arginine, and histidine.

Once the dsDNA immobilization on silicon chips and selective DNA-proteinbinding was verified with stains, the dsDNA and protein detection wasdone on nano-electrode CMOS chip, without any staining. FIG. 18 shows aschematic of the surface bound dsDNA and protein bound to dsDNA betweennano-electrodes. FIG. 20 shows an SEM micrograph of the nano-electrodes.The current-voltage (I-V) measurements were performed using AgilentSemiconductor Parameter Analyzer (4155C) on a probe station. A chipwithout any biomolecules was used a control. The I-V data was recordedfrom −1 V to +1 V across the metal electrodes 500 nm apart. The I-V datashowed linear trend after the capture of proteins on surface immobilizeddsDNA. The yield of devices was 20%, which can be substantiallyincreased by using electrodes with lesser separation or by tagging theprotein molecule with conducting nanoparticles, e.g. of gold. The I-Vdata showed a linear trend indicating conducting behavior of theprotein. The control chip showed open circuit behavior before and afterthe functionalization (triangles in FIG. 19). The resistances of thedevices after protein capture ranged from few ohms to GΩ, indicating avarying number of proteins bridging the gaps between the electrodes.

A CMOS chip is presented, with electronic recognition of selectiveprotein. The selectivity is achieved by using a dsDNA fragment. The I-Vmeasurements are carried out to detect the capture of the protein. Worksdescribing the capture of folate binding protein using antibodies as thecapturing agent report sensitivities such as 130 ng/ml by SurfacePlasmon Resonance; 1.5 ng/ml by Quartz Crystal Microbalance; 5-100 ng/mlby Enzyme-Linked Immunosorbent Assay (ELISA) and 50 pg/ml by opticaldiffraction. Using DNA as the capturing agent, there is a detectioncapability down to 0.28 pmol (less than a pg/ml) of a protein. Suchframework can be easily extended to carry out data acquisition, analysisand decision making on-board the same chip. This new approach is namedProteomic (“protein”+“electronic”) Biochip. With the advent of so called“Omics” revolution, diseases can be defined at both the molecular andthe genomic/protein network levels, and proteomic chips can be used todetect disease linked protein biomarkers to speed up diagnosis andtherapy. The application of the chips could also be extended toenvironmental sample analysis as well, such as in bioterrorism toidentify dangerous virus or bacteria or to identify contaminants in foodand water, etc. Samples from suspect fluids or tissues can beelectrically tested for the presence of important biomolecules.

Example 3

Materials and Methods

A. Materials

All chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) unlessotherwise noted.

B. Aptamer Preparation

The anti-EGFR modified RNA aptamer was isolated by iteratively selectingbinding species against purified human EGFR from a pool that spanned a62 nucleotide random region. A high affinity (K_(d)=2.4 nM) anti-EGFRaptamer and a non-functional, scrambled counterpart were extended with acapture sequence. The capture sequence did not disrupt aptamerstructures but was used as a hybridization handle for binding withprobes immobilized on surface.

The sequences for the extended anti-EGFR aptamer, mutant aptamer, andrelevant capture oligonucleotides were:

anti-EGFR aptamer  (SEQ ID NO: 1)(5′-GGC GCU CCG ACC UUA GUC UCU GUG CCG CUA UAA UGC ACG GAU UUA AUC GCC GUA GAA AAG CAU GUC AAAGCC GGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GAC CAG-3′); mutant aptamer  (SEQ ID NO: 2)(5′-GGC GCU CCG ACC UUA GUC UCU GUU CCC ACA UCA UGC ACA AGG ACA AUU CUG UGC AUC CAA GGA GGA GUU CUC GGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GAC CAG-3′);modified capture oligonucleotide  (SEQ ID NO: 7)(5′-amine-CTG GTC ATG GCG GGC ATT TAA TTC-3′ or  (SEQ ID NO: 8)(5′-6FAM-CTG GTC ATG GCG GGC ATT TAA TTC-3′).

The extension sequence is underlined.

The anti-EGFR aptamer was prepared by transcribing a double-stranded DNAtemplate using Durascribe kits from Epicentre (Madison, Wis.). The DNAtemplate was PCR-amplified, ethanol precipitated, and mixed withreaction buffer, DTT, ATP, GTP, 2′ F-CTP, 2′ F-UTP, and a mutant T7polymerase for 10 hours at 37° C. The DNA template was then degradedwith DNase treatment for 30 min at 37° C. Aptamer was purified on an 8%denaturing PAGE. The band for the aptamer was visualized by UVshadowing, and the aptamer was excised and eluted in 0.3 M NaAc (pH 5.2)overnight at 37° C. followed by ethanol precipitation. The pellet wasdissolved in water, and the concentration of aptamer was measured on aNanoDrop spectrophotometer (Thermo Scientific, Wilmington, Del., USA).The aptamer was modified by extending the DNA template at its 3′ endwith a 24 nt sequence tag, and then hybridizing the transcribed,extended aptamer with a complementary DNA oligonucleotide (referred toas capture oligonucleotide) labeled with 6-FAM or an amine at its 5′end.

C. Preparation of Anti-EGFR Aptamer/Antibody Functionalized Substrates

The glass slides, used as substrates, were cut into 4×4 mm² pieces andcleaned in piranha solution (H₂O₂:H₂SO₄ in a 1:3 ratio) for 10 minutesat 90° C. After rinsing with deionized water (DI water) and drying innitrogen flow, the glass substrates were immersed in a 19:1 (v/v)methanol:DI water solution containing 3% APTMS for 30 minutes at roomtemperature. The substrates were then sequentially rinsed with methanoland DI water and cured at 120° C. for 30 minutes. Silanized substrateswere then immersed in a dimethylformamide (DMF) solution containing 10%pyridine and 1 mM phenyldiisothiocyanate (PDITC) for 2 hours. Eachsubstrate was then washed sequentially with DMF and 1,2-dichloroethaneand dried under a stream of nitrogen. The DNA capture probes with anamine group modification at the 5′ end were prepared at 10 μMconcentration in DI water with 1% (v/v) N,N-diisopropylethylamine(DIPEA). A volume of 5 μl of DNA solution was placed on each substrateand allowed to incubate in a humidity chamber at 37° C. overnight. Eachsubstrate was then sequentially washed with methanol anddiethylpyrocarbonate (DEPC) treated DI water (0.02% v/v). Thefunctionalized surface was then deactivated by capping unreacted PDITCmoieties by immersion in 50 mM 6-amino-1-hexanol and 150 mM DIPEA in DMFfor 5 hours. Each device was then sequentially rinsed with DMF, methanoland DEPC-treated DI water. The incubator was cleaned with RNase-free andDEPC-treated DI water three times. A volume of 5 μl anti-EGFR RNAaptamer at 1 μM concentration was placed on each substrate in 1×annealing buffer (10 mM pH 8.0 Tris, 1 mM pH 8.0 EDTA, 100 mM NaCl).After 2 hours of hybridization at 37° C., substrates were washed with 1×annealing buffer and DEPC treated DI water for 5 minutes. The negativecontrol devices were hybridized with mutant aptamer using the sameprotocol. The substrates were placed in 1× (pH 7.5) phosphate bufferedsaline (PBS) with 5 mM magnesium chloride and kept at −20° C. for oneweek or used immediately. A 100 μg/ml EGFR antibody solution was placedon the glass substrates, and incubated at 37° C. for 1 hour. Then thesubstrates were blocked with BSA (10 mg/ml) solution for 20 min andwashed thoroughly with PBS, and placed in PBS solution.

D. Genetic Engineering, Isolation and Characterization of EGFROver-Expressed Mouse Derived Tumor Cells (Ink4a/Arf−/−EGFRvIII NeuralStem Cells)

Embryonic (E13.5) neural stem cells (NSC) were isolated fromInk4a/Arf−/− embryo brain, maintained under standard neuro sphereculture conditions and were infected with a retrovirus expressing themutant EGFRvIII receptor. The tumorigenicity of these cells have beenextensively characterized. The Ink4a/Arf−/−EGFRvIII NSCs were alsostably transduced with a lentivirus expressing monomeric-cherry(referred to as m-cherry) fluorescent protein, live-cell imaging andidentification.

E. Isolation and Characterization of Human Glioblastoma (hGBM) Cells

Human glioblastoma samples were obtained from consenting patients at theUniversity of Texas Southwestern Medical Center (Dallas, Tex., USA) withthe approval of the Institutional Review Board. On average,specimens >50 mm³ were placed into ice cold hank's buffered saltsolution (HBBS) media immediately upon removal from the brain. Red bloodcells were removed using lympholyte-M (CEDARLANE Labs, Burlington, N.C.,USA). The hGBM tumor tissue was gently dissociated with papain anddispase (both 2%), triturated, and then labeled with a CD133/2(293C3)-PE antibody (Miltenyi Biotec, Auburn, Calif., USA) and sortedwith FACSCalibur machine (BD Biosciences, San Jose, Calif.). Both CD133positive and negative cells were suspended in a chemically definedserum-free Dulbecco's modified Eagle's medium (DMEM)/F-12 medium,consisting of 20 ng/ml mouse EGF (Peprotech, Rocky Hill, N.J., USA), 20ng/ml of bFGF (Peprotech, Rocky Hill, N.J., USA)), 1×B27 supplement(Invitrogen, Carlsbad, Calif., USA), 1× Insulin-Transferrin-Selenium-X(Invitrogen, Carlsbad, Calif., USA), 100 units/ml:100 μg/ml ofPenicillin:Streptomycin (HyClone, Wilmington, Del., USA) and plated at adensity of 3×10⁶ live cells/60 mm plate. Both CD133 positive andnegative fractions underwent clonal expansion and formed orthotopictumors (data not shown). For all the experiments, the CD133 positivefraction, referred to as hGBM cells, was used. The hGBM cells werestably transduced with a lentivirus expressing m-cherry fluorescentprotein.

F. Meninge Derived Primary Fibroblast

Rat derived primary meningeal fibroblasts were obtained from postnatal 3day rat pups. Briefly, meninges were peeled from the cerebral corticesthen processed by incubation for 30 minutes in 0.5% collagenase, 20minutes in 0.06% trypsin/EDTA, and then triturated. Followingtrituration the cells were plated in T-75 tissue culture flasks inDMEM/F-12 medium containing 10% fetal bovine serum and allowed to growfor one week to confluence.

Results

A. Aptamer Binding to Cultured Tumor Cells

To demonstrate the selective binding of aptamer to tumor cells, theanti-EGFR aptamer, annealed with 6-FAM modified capture oligonucleotide,was incubated with tumor cells and fibroblasts, and interaction wasmeasured as follows: The DNA capture probe labeled with 6-FAM was usedas received (Alpha DNA, Montreal, Quebec, Canada). Equal amounts ofanti-EGFR RNA aptamer and DNA capture probe were annealed by heatingsamples to 70° C. for 10 minutes and then slowly cooling to roomtemperature. Both mouse-derived tumor cells and primary fibroblasts wereseeded into separate PDMS wells (8 mm diameter) and cultured for 48hours. The RNA:DNA capture probe was incubated with cells at 37° C. for30 minutes under 5% CO₂. After incubation, the cells were washed with1×PBS 3 times, and stored in fresh sterilized 1×PBS for differentialinterference contrast (DIC) and fluorescence imaging. DIC data was usedto image the cells and fluorescence imaging focused on aptamers. Mutantaptamer was also applied into the cells as a control, and allexperimental procedures were the same as the anti-EGFR aptamer. Thefluorescence images were taken using appropriate filters. The excitationand emission wavelength of 6-FAM is 492 and 517 nm respectively.

B. Tumor Cell Capture Using Anti-EGFR Aptamer/Antibody Substrates

In all experiments, the cell suspensions were centrifuged, thesupernatants were removed, and sterilized and warmed 1×PBS solution(with 5 mM MgCl₂) was added to dilute the cells. About 50 μl of cellsuspension in 1×PBS was placed on each substrate. The substrates wereincubated for 30, 60, or 90 minutes at 37° C., and then washed withsterilized 1×PBS solution on a shaker (Boekel Scientific, Feasterville,Pa., USA) at 90 rpm for 6-10 minutes in orbital and reciprocalmovements. The time of incubation was also studied for saturationeffects. There was no difference seen in the results for the threedifferent groups of 30, 60 and 90 minutes incubation. The bufferevaporation was seen for longer incubation. The subsequent incubationsof cells were thus done for 30 minutes. For tumor specific isolationstudies, the human GBM cells were mixed with fibroblasts in a 1:1 ratio.Mutant aptamer functionalized substrates were used as a control. Theexperiments of EGFR capture with antibodies follow exactly the sameprocedure.

C. Temporal Monitoring of Cellular Interaction Between Tumor Cells andAnti-EGFR Aptamers Functionalized on Substrates

To visualize tumor cell capture via the anti-EGFR aptamer substrates,these were placed on a custom-designed neuro-optical microfluidicplatform, and the interaction between tumor cells and surface graftedaptamers was monitored. Briefly, the substrates were placed on theplatform which maintained 5% CO₂ at 37° C. and high humidity for livecell imaging. The cellular interaction between cells and anti-EGFRaptamer surfaces were closely monitored using an inverted microscope(63×: DIC). Images were taken after every 30 seconds, and theinteraction was monitored for 30 minutes. The tumor cells were alsoseeded on the control substrates (with mutant aptamer) and theinteraction was closely monitored in a similar manner.

D. Quantification and Statistical Analysis

For analysis, 5 representative images were taken from each substrate.The images were analyzed with Image-Pro Plus software. The total numberof captured cells and their relevant diameters on the surface werecounted automatically, and the cell densities were calculated. In orderto show the diameter of tumor cells on aptamer grafted substrates, thedata was sorted into 6 groups based on cell sizes (from 20 μm to themaximum; 5 μm interval) and relevant percentages were obtained.

Discussion

The use of microfluidic devices to isolate rare tumor cells is of greatimportance. The capture of tumor cells requires the affinity recognitionof specific biomarkers. The challenge is to efficiently isolate smallnumber of tumor cells from a much larger pool of normal cells. Aptamersmay prove to be uniquely useful for lab-on-chip devices because of theirhigh and specific affinities for analytes, and the versatility ofconjugation and labeling inherent in their chemical synthesis. Beforethe RNA aptamer substrates to identify and isolate EGFR over-expressedcancer cells were used, the specific binding between mouse-derived tumorcells and the anti-EGFR aptamers was confirmed.

A. Aptamer-Binding to Mouse-Derived Tumor Cells

The capture oligonucleotides modified with 6-FAM dye were hybridized tothe anti-EGFR and mutant (as control) aptamers and specific binding tocultured mouse-derived tumor cells was observed (FIG. 21(a)). Anadditional control for specificity was to incubate the anti-EGFR aptamerwith a non-cognate cell, primary fibroblasts. After washing, greenfluorescence was observed only with the mouse-derived tumor cell surfaceincubated with the labeled anti-EGFR aptamers (FIG. 21(b)). The controlsincluded: (i) mutant aptamer incubated with mouse-derived tumor cells;(ii) anti-EGFR aptamer with fibroblast; and (iii) mutant aptamer withfibroblasts. The fluorescence intensity data are shown in FIG. 21(c).Similar results were obtained for hGBM cells. This showed the specificbinding of anti-EGFR aptamer with tumor cells.

B. Capture and Morphological Characteristics of Mouse Derived TumorCells which Over-Express EGFR

The functionalization of the substrates yields a homo-bifunctional layerof PDITC that can be used to immobilize any amine-modified molecules. Anamine-bearing capture oligonucleotide was conjugated to the surface, andin turn allowed the capture of the extended aptamer. The capping ofun-reacted PDITC end-groups ensured that non-specific adsorption ofaptamer did not occur. The use of capture oligonucleotides for theconstruction of lab-on-chip devices has many advantages: it demonstratesa generalized approach to capture any functional nucleic acid, adistinct advantage relative to the use of proteins; it increases thedistance between the substrate surface and the aptamer, alleviating theeffects of steric and/or electrostatic hindrance that may come fromsurface tethering; and it increases the radius of gyration of theaptamer, thereby potentially increasing reactivity. This also resultedinto very distinct behavior of cells when interacting with aptamers(discussed later).

Genetically engineered mouse glioma cells were incubated on the aptamersubstrates and washed with warmed 1×PBS (FIG. 22). Significantly highnumbers of the mouse-derived EGFR over-expressed tumor cells were seenbound to the anti-EGFR aptamer functionalized surfaces (Avg: 392cells/mm², Max: 831 cells/mm², Min: 284 cells/mm², S.D.: 143.3), with anisolation efficiency of 62.32%. Almost none of the cells were capturedon the mutant aptamer functionalized control substrates (Avg: 7cells/mm², Max: 11 cells/mm², Min: 0 cells/mm², S.D.: 2.8) (FIG. 3).

These results reveal an additional, important feature of the use ofnucleic acids on lab-on-chip devices. Nucleic acids may provide apassivation layer that minimizes non-specific adsorption. Thehydrophilic surface and electrostatic repulsion may have prevented anynon-selective physical adsorption of the cells on mutant aptamersubstrates. The density and amount of sialylation on cancer cellssurface is known to be higher than normal ones. Carboxyl groups fromsialic acid cause a net negative surface charge on cancer cells. Therepulsion between negatively charged cells and the negative charges fromthe surface functionalized aptamer can be another putative reason forthe lack of non-specific adsorption. On the other hand, the cancer cellsthat could selectively interact and bind to the aptamer got captured onthe surface, even in the presence of above-stated competing forces. Theuse of capture oligonucleotides thus has the advantage of reducingnon-specific binding or adsorption, adding to the selectivity of thesubstrates. The use of DNA to covalently immobilize aptamers thusprovides a robust passivation of the surface that provides functionalityand selectivity while screening effects of surface charges.

The density and diameters of captured cells showed distinct behavior onthe anti-EGFR and mutant aptamer substrates (FIG. 23). On average therewere ˜392 cancer cells captured per mm² on 12 anti-EGFR aptamersubstrates (S.D: 143.3), with the size ranges depicted in FIG. 23(b).Interestingly ˜70% of the captured cells had diameters above 30 μm,whereas the size of these cells in suspension ranged between 25-30 μm.This indicates that cancer cells were spreading on the anti-EGFR aptamersubstrates. This can serve as a novel and important phenomenon that canbe a discriminating factor in cytological studies for the confirmationof the captured tumor cells.

C. Capture of hGBM Cells

EGFR expression level on hGBM cells was lower (approximately 50%)compared to the genetically mouse glioma cells (level verified bywestern blot, data not shown). Despite the relative lower EGFRexpression level, large number of hGBM cells was captured by theanti-EGFR aptamer functionalized substrates. These cells did not bind tomutant aptamer control substrates (FIG. 24).

FIG. 24 shows the clear difference in the number and cell shapes of hGBMcells on anti-EGFR and mutant aptamer substrates. Along with differencein the numbers of captured cells, the shapes of the cells bound withaptamers and adsorbed on mutant aptamer surfaces were also quitedifferent (discussed later). Analysis of 12 substrates showed that onaverage 117 hGBM cells were captured per mm² on anti-EGFR aptamersubstrate (S.D: 44.4, max and min of 228 and 56 cells/mm²,respectively), with isolation efficiency of 38.74%. In the controlmutant substrate group average density of 4 cells/mm² was seen (S.D:4.1, max and min densities of 13 and 0 cells/mm² respectively). Incomparison to the mouse derived tumor cells, fewer number of capturedhGBM cells than that for mouse-derived tumor cells (discussed inprevious section) can be explained in terms of overly high number ofEFGR that were genetically engineered in mouse derived tumor cells. Thedecreased density of captured hGBM cells was thus as expected.

D. Isolation of Cancer Cells from Cell Mixture

In above two experiments (mouse-derived tumor cells and primary hGBMcells), it was confirmed that the anti-EGFR aptamer functionalizedsubstrates could capture significantly more tumor cells as compared tothat with the mutant aptamer. Towards the application of aptamerfunctionalized substrates in isolating tumor cells and study theirbehavior, a cell mixture was used. A mixture of hGBM and fibroblastcells was prepared in a ratio of 1:1. The substrates were incubated inthe mixture, washed and the results were imaged. In parallelexperiments, only hGBM cells were incubated with substrates. Both DICand fluorescent images were taken (FIGS. 25(a) and 25(b)). In hGBM-onlysurfaces, the fluorescent intensities were not uniform when DIC andfluorescent images were overlaid (hGBM cells were modified to expressedm-cherry fluorescent protein for clear differentiation). There wereabout 16.7% cells from 12 substrates (189 out of 1133 cells, Average:˜16, S.D: 5.1) that did not show any fluorescence. The data from themixture group showed no fluorescence from about 27.5% cells from 12substrates (378 out of 1376 cells, Average: 31.5, S.D: 6.8). The cellsthat did not show up in fluorescence images included captured hGBM andnon-specifically bound fibroblast cells. The difference of the twopercentages, as a first order approximation, shows that on average about10.8% captured cells were fibroblasts. Thus the anti-EGFR aptamersubstrates can selectively isolate and enrich a 1:1 mixture suspensionof fibroblasts and cancer cells to 1:8.24 on the surface. In EGFRantibody substrates control group, the ratio of captured fibroblasts andcancer cells was 1:2.77. The specificity of aptamer and antibody oncancer cell isolation was thus 94.82% and 68.81% respectively. Theresults show aptamer has higher specificity. In a practical scenario, asa lower limit, the aptamer grafted substrates can enrich the amount ofcancer cells by an order from the concentration in the solution. In acyclic iteration application, a sample can be run for multiple timesover the substrates to increase the capture efficiency. It may beimportant to note here that “mean capture yield” using Anti-EpCAMantibodies has been shown to be ˜65%.

E. Shape and Size of Cancer Cells on Functionalized Substrates

In all the experiments, the cell shapes and sizes showed a distinctbehavior: fibroblast altered their fusiform, stellar or irregular shapeto spherical. The fibroblast cells have normal EGFR expression on theircell membrane but the amount of EGFRs are far less than those for hGBMor mouse derived tumor cells. To bind with anti-EGFR aptamer the cellsaltered their shape to decrease their surface area to increase the EGFRdensity that would come in contact with the surface bound aptamer. Thetemporal images of mouse-derived tumor cells also showed changes in cellshapes from spherical in suspension to semi-elliptical and flat on theaptamer-grafted surfaces. The possible reason between different cellbehavior on surface maybe a result of different elasticity that is knownto be different in cancer cells. The EGFR over-expressed cells werere-shaping to cover as large of an area as possible. Temporal imagingalso showed tumor cell growth and lots of activity on surfaces. FIGS.26(a) and 26(c) were taken at the beginning when mouse-derived tumorcells were seeded on the anti-EGFR and mutant aptamer surfaces, andFIGS. 26(c) and 26(d) were taken after 30 minutes. Changes in cellsshapes and flatness are evident in FIGS. 26(a) to 26(c) and from 26(b)to 26(d). The size of cells also became bigger, and many antennae formedduring the incubation period on anti-EGFR aptamer substrates. The datashows that the tumor cells on the substrate surface had strong activityand were arbitrariliy changing their shape. In contrast, in FIGS. 26(c)and 26(d), where first, only much fewer cells did get capture on themutant aptamer surfaces, and second, those too faced repulsion from thehydrophilic glass surface and the negative charges from the immobilizedoligonucleotides. As a result, the cells had almost no change in theirsizes during the 30 minutes. The data on hGBM cell shapes in FIG. 26(b)also show that cells on mutant aptamer substrates maintained globularshape as discussed above. The spreading and flatness of cancer cells onaptamer surfaces can be an important modality for detection, as anadditional method to support histological findings and further identifytumor cells based on their physical behaviors. In addition, hGBM cellsare diffusively infiltrative and current methods to define tumor marginsfor surgical resection, using MR imaging, are inadequate. Histologicalevidence suggests that tumor cells at the leading edge may express highlevels of EGFR. It is possible, therefore, that freshly resected tumorcould be enzymatically dissociated and captured on the anti-EGFR aptamerfunctionalized substrates, in real time, to better define tumor margins.Such information, thus, can help guide the extent of tumor resection aswell. There is considerable evidence that the extent of resection isdirectly related to overall survival. Beyond the specific applicationfor management of hGBM tumors, our findings are especially importantgiven that enrichment of rare CTCs may be difficult for virtually anylab-on-chip device. The use of aptamers leads both to high passivationand the presentation of unique physical morphologies, and thus may be anovel first level detection step in point-of-care examination of CTCs.

Conclusions

It has been demonstrated that anti-EGFR RNA aptamer substrates canspecifically recognize, capture and isolate cancer cells that are knownto over-express EGFR. The aptamers selectively captured mouse derivedtumor cells which after genetic modification over-expressed EGFR on thecell membrane. Aptamer substrates also specifically isolated human GBMcells from a mixture of fibroblasts. The isolation efficiency dependedon strong binding between aptamer and the amount of EGFR expression onthe cell membrane. The change in cell shape and cellular activity canserve as a novel way of identifying tumor cells. The substrates can alsobe used for identification and isolation of CTCs from peripheral blood,dramatically changing intervention and prognosis of metastasis.

Example 4

Materials and Methods

A. Materials

All chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) unlessotherwise noted.

B. Aptamer Preparation

Purified human EGFR (R&D systems, Minneapolis, Minn.) was used foranti-EGFR RNA aptamer preparation via selecting binding species. TheEGFR protein was purified from murine myeloma cells, and contained theextracellular domain of human EGFR (Leu25-Ser645) fused to the Fc domainof human lgG1 (Pro 100-Lys 330) via a peptide linker (IEGRMD). Theanti-EGFR aptamer (K_(d)=2.4 nM) and a mutant aptamer were extended witha capture sequence. The extended capture sequence did not participate inaptamer hairpin structure but was used to immobilize aptamer on thesubstrate through duplex formation with substrate anchored probe. Thesequences of the extended anti-EGFR aptamer, extended mutant aptamer,and substrate anchored probe were:

anti-EGFR aptamer (SEQ ID NO: 3)(5′-GGC GCU CCG ACC UUA GUC UCU GUG CCG CUA UAA UGC ACG GAU UUA AUC GCC GUA GAA AAG CAU GUC AAA GCC GGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GAC CAG-3′); mutant aptamer  (SEQ ID NO: 4)(5′-GGC GCU CCG ACC UUA GUC UCU GUU CCC ACA UCA UGC ACA AGG ACA AUU CUG UGC AUC CAA GGA GGA GUU CUC GGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GAC CAG-3′);substrate anchored probe  (SEQ ID NO: 5)(5′-amine-CTG GTC ATG GCG GGC ATT TAA TTC-3′).The extended capture sequence is underlined. The aptamer was modified byextending the DNA template at its 3′ end with a 24 nucleotide sequencetag, and then hybridizing the transcribed, extended aptamer with acomplementary substrate anchored probe modified with an amine at its 5′end.C. Preparation of Nano-Textured PDMS Substrates

Half gram of poly(lactic acid)/poly(glycolic acid) (PLGA; 50/50 wt %;12-16.5×10³ MW; Polysciences, Inc.) was dissolved in 8 ml of chloroformat 55° C. for 40 minutes. The solution was cast into glass petri dishes,allowed to sit overnight, and was put into a vacuum chamber (15 in Hg)for 2 days at room temperature. The solid PLGA polymers were treatedwith 10 N NaOH for 1 h to generate nanostructured surfaces, and furthersterilized by soaking in ethanol for 24 h followed by exposure to UVlight for 1 hour. SYLGARD 184 Silicon Elastomer (Dow Corning, Midland,Mich.) was mixed (10:1, wt/wt) with a silicon resin curing agent. Themixture was first placed in a vacuum chamber to remove all bubbles. Theelastomer was then cast onto NaOH treated PLGA polymer surface, and wasthen allowed to cure for 48 h at room temperature. Finally, the PDMS waspeeled from the PLGA. Before the surface modification, the PDMSsubstrates were immersed into DI water at 37° C. overnight to completelyremove any residual PLGA.

D. SEM and AFM Characterization

Zeiss Supra 55 VP scanning electron microscope was used to qualitativelyevaluate PDMS surface topography. Samples were sputter-coated with goldat room temperature and visualized at 100× magnification at 5 kVacceleration voltage. Surface topography was quantitatively evaluatedusing Dimension 5000 AFM. The changes in surface area and root meansquare surface roughness were measured. Height images of PDMS sampleswere captured in the ambient air at 15-20% humidity at a tappingfrequency of approximately 300 kHz. The analyzed field was 3 μm×3 μm ata scan rate of 1 Hz and 256 scanning lines.

E. Attachment of Anti-EGFR Aptamer on PDMS and Glass Substrates

The PDMS substrates and the glass slides were cut into 5×5 mm² piecesand cleaned with UV Ozone plasma and piranha solution (H₂O₂:H₂SO₄ in a1:3 ratio) for 30 and 10 minutes respectively. After rinsing withdeionized (DI) water and drying in nitrogen flow, the PDMS and glasssubstrates were immersed in 2% (v/v) of APTES and methanol solution for30 min at room temperature. The substrates were then sequentially rinsedwith methanol and DI water. The amino groups on PDMS and glasssubstrates were converted to the isothiocyanate groups by introducing a0.5% (v/v) thiophosgene solution in acetonitrile for 20 min at 40° C.The substrates were then washed with DI water and dried in a stream ofnitrogen. The amino modified DNA capture probes were prepared at 30 μMconcentration in 5 mM tris buffer with 50 mM NaCl. A volume of 5 μl ofDNA solution was placed on each substrate and allowed to incubate in ahumidity chamber at 37° C. overnight. Each substrate was then washedwith DI water. Salmon sperm DNA was used for prehybridization to reduceRNA physical adsorption. A volume of 5 μl anti-EGFR RNA aptamer at 1 μMconcentration was placed on each substrate in 1× annealing buffer (10 mMpH 8.0 Tris, 1 mM pH 8.0 EDTA, 100 mM NaCl). After 1 hour ofhybridization at 37° C., substrates were washed with 1× annealing bufferand DI water for 5 minutes. The negative control devices were hybridizedwith mutant aptamer using the same protocol. The substrates were placedin 1× (pH 7.5) phosphate buffered saline (PBS) with 5 mM magnesiumchloride and used immediately.

F. Contact Angle Measurements

Contact angles were measured on isothiocyanate group modified PDMS(with/without nano-texturing), unmodified PDMS (with/withoutnano-texturing), and glass (with/without isothiocyanate groupsmodification). A droplet of DI water was placed on the surface of thesubstrate at room temperature, and after 30 s, the contact angle wasmeasured using a contact angle goniometer (NRL-100, Rame-Hart). Averageof five measurements were utilized for each droplet.

G. Fluorescence Measurements of Fluorescamine

Surface modification was further confirmed by fluorescence measurementsof fluorescamine. The density of surface-grafted amino groups from APTESwas measured by fluorogenic derivatization reaction with fluorescamine.A mixture of 900 μl of 0.1% (w/v) fluorescamine dissolved in acetone,150 μl of 0.1 M borate buffer and 1.91 ml DI water was made. After APTESmodification, glass, PDMS and nanostructured PDMS samples were immersedinto fluorescamine mixture solution for 5 min at room temperature. Allsamples were rinsed with acetone to remove the excessive reagents. Thefluorescence measurements were taken at 390 nm wavelength using ZeissConfocal Microscope. The fluorescence intensities were analyzed withImageJ software.

H. Human Glioblastoma and Meninge Derived Primary Fibroblast CellsCulture

The hGBM cells were cultured in a chemically defined serum-free medium:Dulbecco's modified Eagle's (DMEM)/F-12 medium supplemented with, 20ng/ml mouse EGF (Peprotech, Rocky Hill, N.J., USA), 20 ng/ml of bFGF(Peprotech, Rocky Hill, N.J., USA)), 1×B27 supplement (Invitrogen,Carlsbad, Calif., USA), 1× Insulin-Transferrin-Selenium-× (Invitrogen,Carlsbad, Calif., USA), Penicillin:Streptomycin (100 units/ml:100 μg/ml)(HyClone, Wilmington, Del., USA) and plated at a density of 3×10⁶ livecells/60 mm plate. The hGBM cells were stably transduced with alentivirus expressing m-cherry fluorescent protein. The primary ratmeninge derived fibroblasts were plated in T-75 tissue culture flasks inDMEM/F-12 medium containing 10% fetal bovine serum.

I. Tumor Cell Capture on Substrates

The cell suspensions were centrifuged, the supernatants were removed.Sterilized and warmed 1×PBS solution (with 5 mM MgCl₂) was added todilute the cells. About 500 μl of cell suspension in 1×PBS was placed oneach substrate surface. The substrates were incubated for 30 minutes at37° C., and then washed with sterilized 1×PBS on a shaker at 90 rpm for15 minutes. For tumor specific isolation studies, the GBM cells weremixed with fibroblasts in a 1:1 ratio.

Results and Discussion

A. Surface Topography of Nano-Textured Polymers

The effects of NaOH concentration and etching times have been previouslycharacterized before. PLGA bulk surface was etched with 10 N NaOH for 1h to generate nano-textured surface. SEM images of PLGA before and afterNaOH etching showed different surface properties (FIG. 27). Theuntreated one (insert to FIG. 27) was smoother than the nano-texturedsurface. AFM was used to quantitatively analyze the average surfaceroughness. AFM micrographs of PLGA substrate showed that NaOH treatmentresulted into nano-textured surfaces with an increase in surfaceroughness and surface area (FIG. 28). Surface roughness increased from22 nm on untreated PLGA surface to 310 nm on nano structured PLGAsurface.

The silanization and isothiocyanate molecule incubations were done for30 and 20 min respectively. It is important to not to incubate the PDMSsubstrates for longer periods, as that may cause PDMS bulk to dissolveand swell. Solubility parameters of ethanol and acetone are 12.7 and 9.9cal^(1/2) cm^(−3/2) respectively, and solvents that have a solubilityparameter similar to that of PDMS (7.3 cal^(1/2) cm^(−3/2)) generallyswell PDMS more. Solubility parameters of methanol and acetonitrile are14.5 and 11.9 cal^(1/2) cm^(−3/2) respectively, and the swelling ratiosare 1.02 and 1.01 respectively, less than that of ethanol and acetone(1.06 and 1.04 respectively). That is why methanol and acetonitrile wereused for surface modification. Further, although methanol andacetonitrile can completely dissolve PDMS, the process takes extremelylong time. Even diisopropylamine, which has swelling ratio as high as2.13, still need one month to completely dissolve the PDMS. So in theseexperiments, the swelling and dissolving factors were insignificant.After the surface modification, the surface roughness did not show anysignificant changes.

B. Contact Angle Measurements

The contact angle data of a water droplet is frequently used as ameasure of the hydrophobicity of a surface. The contact angles of eachsubstratem were measured. The average (n=10) contact angles and standarddeviations are shown in TABLE III.

TABLE III Contact angle measured immediately following substrate UVozone treatment and chemical activation with PDITC, for each of thesubstrates employed in this study. Substrate Type Base Substrate with -Glass  46° ± 1° 51° ± 1° PDMS No Nano- 11Table 2. Fluorescence 59° ± 2°intensity (a.u.) for glass, PDMS PDMS with 144° ± 4° 46° ± 3°

After APTES and isothiocyanate group modification, all three types ofsubstrates showed hydrophilic surfaces. The aptamer immobilization wouldfurther decrease the contact angle and making these substrates morehydrophilic. The hydrophilic surfaces are known to have lower proteinand cell physical adsorption.

PDMS initially has methyl groups on both side of backbone; after UVozone treatment, the methyl groups are substituted with hydroxyl groups.The residual methyl groups on the PDMS surface still contribute to thehydrophobicity, as a result, the PDMS surface contact angle is higherthan that of glass surface even after APTES and isothiocyanate groupmodification (TABLE III). Due to increased surface roughness, thenano-textured PDMS substrate has the largest contact angle, but thisdecreased to a lowest number after APTES and isothiocyanate modification(from 144° to 46°). Due to higher surface roughness the standarddeviation of nano-textured PDMS is also higher than that of othergroups.

C. Fluorescence Measurements

Fluorescamine is intrinsically non-fluorescent, but its reaction withamino groups results into highly fluorescent derivatives. The relativeamount of amino groups on different samples can be determined bycomparing the fluorescence intensities of each sample. The averagefluorescence intensities of three types of samples are shown in TABLEIV.

TABLE IV Fluorescence intensity (a.u.) for glass, PDMS andnanostructured PDMS after APTES & fluorescamine modification SubstrateType Fluorescence Intensity (a.u.) Glass 4.7 ± 1.5 PDMS withoutNanostructure 52.7 ± 6.3  PDMS with Nanostructure 83.9 ± 14.1

Nano-textured PDMS shows the highest intensity. On a PDMS chain, twomethyl groups can be replaced with hydroxyl groups during the plasmatreatment, and the nano-texturing of the surface increases the effectivesurface area. As a result, the exposure of the surface of PDMS to UVozone can generate a significantly higher number of hydroxyl groupscompared to that on the plain PDMS or glass surface, and so, more aminogroups can be introduced on the surface after silanization treatment.The amino group concentration has been shown to reach 4×10⁻⁸ mol/cm²,and UV ozone treatment can gradually increase the oxygen contentpercentage (almost 60%) with prolonged treatment. In brief, properoxidization of PDMS surface can significantly increase the number ofhydroxyl groups, and further increase the number of available aminogroups from APTES and finally improve the total number of immobilizedaptamers. Increased number of available aptamers on the surface isfavorable for tumor cell isolation. The substrate anchored DNA probedensity can be around 1 per 4-5 nm² on plane substrates, and higherprobe density would decrease the space between 2 adjacent probes. Thenegative charges from the oligonucleotide functionalized surfacesfurther inhibit the aptamers from inserting into the spaces betweenprobe molecules and reduce non-covalent adsorption on the surface.However, on nano-textured PDMS surfaces, the larger effective surfacearea increases the total number of aptamers and the density. It is worthnoting that long UV ozone treatment (over 90 min) makes the PDMS stifferand creates lots of tiny cracks on the surface. In this experiment, thePDMS surfaces were treated for just 30 minutes with UV ozone.

D. Isolation of hGBM Cells

FIG. 29 (A) to (F) depict representative images of the hGBM cellscaptured on glass, PDMS and nano-textured PDMS substrate with anti-EGFRor mutant aptamers. The average density of cells on substrates beforewashing was 400.9 per mm² (S.D.: 43.3). All substrates were washed with1×PBS at 90 rpm for 15 min. Fluorescent images of cells on 10 substratesof each type were taken. The results are shown in FIG. 29 (G) plot. Onaverage 149.6 hGBM cells were captured per mm² on anti-EGFR aptamermodified nano-textured PDMS substrate (S.D.: 12.2). On the other hand,79.3 cells per mm² (S.D.: 11.5) and 37.4 per mm² (S.D.: 10.1) werecaptured on anti-EGFR aptamer modified glass and PDMS substratesrespectively. There are four major factors which influence the cellcapture: the available number of anti-EGFR aptamer molecules on thesubstrate; the EGFR density on the cell membrane; the affinity betweenthe EGFR and aptamer; and the surface quality of the substrate. It isseen that available number of aptamer molecules is a direct function ofsurface nano-texturing. Cell isolation efficacy can be improved byincreasing the affinity between surface bound aptamer and theover-expressed EGFR. In this case the higher affinity comes fromnano-texturing which increases the quantity of aptamers on the surface.The fluorescamine analysis has demonstrated that the nano-textured PDMScan generate more hydroxyl groups after oxidization and therefore moreamino groups from APTES can be attached on the surface aftersilanization. Thus, the density of immobilized anti-EGFR aptamer isincreased. In addition, the nano-textured surface mimics the basemembrane which facilitates cancer cell attachment. Thus, the number ofcaptured cell on the nano-textured PDMS substrate is higher than othertwo groups. As discussed before, flat PDMS surface can also generatemore hydroxyl groups after oxidization, and a few nanometer roughstructuring can be achieved with long UV ozone treatment. However, onflat PDMS surface, even after chemical functionalization, it is still amajor challenge to maintain cells on the surface, especially inlong-term cell culture on PDMS, because stable cell-adhesive layer isnot easy to form. Moreover, the generated hydroxyl groups undergodehydration reaction and reform Si—O—Si bonds, and the high chainmobility pulls the hydrophobic methyl groups to the surface as well.These two factors can prohibit stable cell-adhesive layer formation.Thus the number of captured cells on PDMS surface is lower than that onglass and nano-textured PDMS surface. This can happen on nano-texturedPDMS substrate also, however, the nano-texturing itself provides atrade-off by improving cell attachment and isolation. As the data shows,nano-textured surfaces show improved cancer cell isolation, but thenon-specific cell attachment also increases. Increased surface areaprovides more sites not only for protein adsorption but also for focalcontact adhesion sites used by cells to attach onto the surfaces. Thenano-textured surfaces are thus better suited for overall cell adhesiongoals. In the control group, cell density on mutant aptamer modifiednano-textured PDMS substrate is 25.6 per mm² (S.D.: 6.2), almost 12times higher than that on glass substrate. Obviously, the higherphysical absorption decreases the isolation specificity, but it alsosignificantly improves the detection sensitivity. In practicalapplications, the selection of material and surface structure depends onthe competing goals of isolation sensitivity and specificity. FIG. 30shows the captured cells on the PDMS and nano-textured PDMS surface.After 30 min incubation, hGBM cells can form pseudopods that indicatecells can firmly attach on the nano-textured PDMS surfaces. The samephenomenon is not seen on smooth PDMS surfaces.

F. Isolation of hGBM Cells from Cell Mixture

A mixture of hGBM and fibroblast cells was prepared in a ratio of 1:1,the average cell density on the surface was 332.3 per mm² (S.D.: 23.6).The substrates were incubated in the cell mixture, washed and imaged.Both DIC and fluorescent images were taken (FIGS. 31(a) and 31(b)).There were about 18.4% hGBM cells from 10 substrates (S.D: 9.1) that didnot show any fluorescence. The data from the mixture group showed nofluorescence from about 31% cells from 10 substrates (846 out of 2729cells, Average: 58.8 per mm², S.D: 21.4). The cells that did not show upin fluorescence images included captured hGBM and non-specifically boundfibroblast cells. The difference of the two percentages, as a firstorder approximation, shows that on average about 15.4% captured cellswere fibroblasts. Thus the anti-EGFR aptamer substrates can selectivelyisolate and enrich a 1:1 mixture suspension of fibroblasts and cancercells to 1:5.5 on the surface. In comparison to the previous work onsmooth glass substrate, the ratio of hGBM and fibroblast decreases from1:8.24 (for glass) to 1:5.5 (for nano-textured PDMS). It indicates thenano-textured PDMS substrates also lead to more fibroblast cellsattachment. In other works, decreased fibroblast adhesion onnano-textured PLGA substrate after culture was observed. The decreasedattachment rate of vitronectin on more hydrophilic NaOH-treated PLGAsurface was considered as the primary factor, because thevitronectin—rich PLGA surface is not favorable for fibroblast adhesion.Here, the surface with anti-EGFR aptamer was modified and the mixturecells was incubated for 30 minutes. In other words, it is not believedthat vitronectin can effectively attach to the surface in short time andthus further decrease in fibroblast adsorption was expected. Theincreased number of fibroblasts could then be attributable to thenano-texturing and higher number of available aptamers on the surfacewhich would also bind to the EGFR on fibroblasts' surfaces. Although thenano-textured PDMS substrate increased the attachment of fibroblast, inany case it still could specifically capture hGBM and improve the ratiofrom 1:1 to 1:5.5. The increased sensitivity decreases its specificitybut the trade-off is to the advantage of isolating as many of the smallnumber of cancer cells as possible. In practical applications, theselection of material and surface structure depends on the goals ofisolation sensitivity or specificity.

Conclusions

It is demonstrated that anti-EGFR RNA aptamer modified nano-texturedPDMS substrates can capture more hGBM cells compared to traditionalsmooth glass substrates; moreover, the nano-textured PDMS substrate canstill specifically recognize, capture and isolate hGBM cells from amixture of fibroblasts. The nano-textured surface simulates the basementmembrane structure and can facilitate tumor cell isolation. This canhave important implications for chip-based cancer cell isolationsubstrate selection.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations, and subcombinations of ranges specific embodiments thereinare intended to be included.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in its entirety.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

Example 5

In this Example, a cytological approach to identify cancer cells basedon their dynamic behavior on functionalized surfaces is presented.Aptamers were used to selectively isolate human glioblastoma (hGBM)tumor cells on functionalized substrates (FIG. 32). The hGBM cells areknown to overexpress epidermal growth factor receptors (EGFR) and thesewere specifically distinguished and isolated using anti-EGFR RNAaptamer. During the capture of hGBM cells by the surface-bound aptamers,the cells showed distinct morphological attributes, which were absent innormal cells. Several feature vectors were extracted based on transientmorphological changes from the acquired temporal images duringincubation. The comparison of the vectors from healthy and diseasedcells showed distinctions between tumor and normal cells on the surface.

Methods

The hGBM cells and astrocytes were obtained from consenting patients atthe University of Texas Southwestern Medical Center at Dallas, Tex. asper the approved Institutional Review Board (IRB) protocols. Astrocytesare glial cells from the same lineage as hGBM cells. Therefore,comparing hGBM with astrocytes for differentiating between tumor andnormal healthy cells gives an accurate and precise comparison, whileminimizing other factors that may affect results. Silicon dioxide(glass) surfaces were functionalized with RNA aptamers known toselectively bind to EGFR. Once cells were incubated, the cells gotcaptured. Time lapsed images were taken using an optical microscope andrecorded images were analyzed using software described further below.Conventional image processing along with contour detection were used tofollow cell behavior. Quantitative data was extracted to compare betweencontrol and cancer cells.

A. Selection of Target: Specificity, Selectivity And Sensitivity

EGFRs are overexpressed on tumor cell surfaces with density ranging from40,000 to 100,000 per cell. This upregulation of EGFR has beenassociated especially with lung cancer and glioblastoma. Anothermutation of EGFR, known as EGFRvIII, has also been observed in lung andglio-carcinomas which was found responsible for cells' proliferativenature. Mutant EGFRvIII results in constant activation of the receptorcausing the cells to undergo constant division and thus predisposing theindividual to cancer. The EGFRvIII is characterized by a sequencedeletion of exon 2-7 (amino acid 6-273). Both types of EGFRs are presenton a tumor cell surface but the expression level and the density of thewild type EGFR is much lower (40,000 per cell) than the density ofmutant EGFR (approximately a million per cell). It has been shown thatanti-EGFR RNA aptamer binds specifically with both mouse derived wildtype EGFR and mutant EGFRvIII. In fluorescence experiments that weredone for testing the binding ability of the aptamer to EGFR and itsvariant, mouse glioma cells which were bound to anti-EGFR aptamer showedincreased fluorescent activity (60 a.u.) when compared to fibroblastcells (5 a.u.).

A significant amount of mouse-derived tumor cells were captured withanti-EGFR aptamer modified capture substrate. The anti EGFR aptamers hada capturing efficiency (ratio of the number of captured cells to thetotal number of tumor cells) of 62%. When aptamer and antibody basedtumor cell isolations were compared, it was observed that aptamers hadhigher specificity to tumor cells as compared to the antibodies. It hasbeen shown that the specificity of aptamers on tumor cell capturing is94.82% as compared to antibody's 68.81%. This specificity of the aptamercan be attributed to its ability to bind to the extracellularligand-binding domain III of the receptor which is present in both wildtype and mutant EGFR. The specific binding of the aptamers to the domainminimizes any chances of non-specific adsorption. Tumor cells have highdensity of sialylation on their surface as compared to surfaces ofnormal cells. Increased sialylation gives the tumor cell a net negativecharge which is then intuitively repelled by the negative charge of theaptamer, further minimizing non-specific adsorption.

Aptamers selectivity and sensitivity was further improved by usingcapture oligonucleotide. The capture DNA covalently immobilizes theaptamer on the surface (functionalization). This DNA captures theaptamer, increases the distance between the aptamers and the substratesurface and minimizes any steric and electrostatic hindrance that mayarise due to surface tethering. This also increases aptamer's radius ofgyration, allowing increased reactivity.

B. Aptamer Preparation

The anti-EGFR aptamer sequence used in this Example was: 5′-GGC GCU CCGACC UUA GUC UCU GUG CCG CUA UAA UGC ACG GAU UUA AUC GCC GUA GAA AAG CAUGUC AAA GCC GGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GACCAG-3′. The sequence for mutant aptamer was 5′-GGC GCU CCG ACC UUA GUCUCU GUU CCC ACA UCA UGC ACA AGG ACA AUU CUG UGC AUC CAA GGA GGA GUU CUCGGA ACC GUG UAG CAC AGC AGA GAA UUA AAU GCC CGC CAU GAC CAG-3′; (theextended sequence used to bind to capture DNA is shown in italics).Substrate-anchored capture DNA probe had the sequence: 5′-amine-CTG GTCATG GCG GGC ATT TAA TTC-3′.

C. Preparation of Anti-EGFR Aptamer Functionalized Substrates

Briefly, the aptamer binding protocol was as follows. First, glassslides were used as substrates and were cut into ˜5×5 mm² pieces.Piranha solution (H₂O₂:H₂SO₄, 1:3) was used to clean the slides for 10minutes. The slides were then rinsed with deionized (DI) water and driedin gentle nitrogen gas blow. After drying, the slides were placed for 30minutes in methanol/DI water (19:1) and 3% APTMS solution. The glasssubstrates were then washed with DI water and methanol and incubated for30 minutes at 120° C. A dimethylformamide (DMF) solution was thenprepared using 10% pyridine and 1 mmol/L p-Phenylene diisothiocyanate(PDITC). The substrates were then immersed in the DMF solution for 5hours at 45° C. The substrates were successively rinsed with DMF and1,2-dichloroethane. After rinsing, the slides were then dried withnitrogen gas. A 30 μmol/L of capture DNA solution with a 5′ end aminegroup was prepared using DI water with 1% N,N-Diisopropylethylamine(DIPEA). The 15 μL of the DNA solution was then placed on each glasssubstrate. The substrate was then incubated overnight in a humid chamberat 37° C. After incubation the substrates were successively washed withmethanol and diethylpyrocarbonate (DEPC) treated DI water. To preventany non-specific protein adsorption, unreacted PDITC moieties were thencapped to deactivate the functional surfaces. This was done by immersingthe glass slides for 5 hours in 150 mmol/L DIPEA in DMF and 50 mmol/L6-amino-1-hexanol. Again, each substrate was sequentially washed withethanol, DMF, and DEPC-treated DI water. RNase free and DEPC treated DIwater was used to properly wash the incubator. In the incubator, a 5 μlof 1 μmol/L anti-EGFR RNA aptamer was placed on each substrate in thepresence of 1× annealing buffer [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA(pH 8.0), 1 mmol/L NaCl]. After being subjected to 2 hours ofhybridization at 37° C., substrates were then washed with 1× annealingbuffer and DEPC-treated DI water for 5 minutes. A mutant aptamer usingthe same protocol was hybridized onto control substrates and used as anegative control device. Substrates were then used immediately or storedin 1×PBS (pH 7.5) with 5 mmol/L magnesium chloride solution until used.

D. Fluorescence Measurement

Immobilization of ssDNA and RNA aptamers on the glass surface wasverified by fluorescence measurements of Acridine Orange (AO) stain atan excitation wavelength of 460 nm and an emission wavelength of 650 nm.The chip surfaces were prepared as described above and stained atdifferent immobilization steps. In short, AO solution of concentration 2mg/ml was prepared in sterilized DI water and the samples werecompletely immersed into it and kept on the shaker for 30 minutes. Itwas later washed thoroughly with DEPC water before fluorescencemeasurement. The fluorescence intensity was analyzed with ImageJsoftware.

E. Isolation and Characterization of hGBM Cells

The hGBM cells were readily placed in ice cold HBSS solution after beingtaken from the patient's brain. The specimens were, on average, largerthan 50 mm³. Lymphocyte-M (Cedarlane labs) was used to remove the redblood cells from the specimen. A solution of 2% papain and dispase wasused to gently dissociate the intact hGBM cells, followed by gentlegrinding (trituration). A FACS Calibur machine (BD Biosciences) was thenused to sort out the cells. Clonal expansion and formation of orthotopictumors was observed in both CD133+ and CD133− fractions. Cells from theCD133+ fraction were then used in the experiments.

F. Image Processing

Time lapsed optical micrographs were acquired at 30 second intervalsusing a Leica microscope with DFC295 color camera at 20× magnification.A moving stage microscope was used to image the entire chip. Celldensity was measured using a hemocytometer and was optimized at 100,000cells per milliliter to avoid cells attaching to each other. The imageswere saved at 4096×3072 resolutions in tiff format.

G. Image Segmentation

Each cell was cropped out using an image segmentation algorithm. Thealgorithm provided an approximation of the center of the cell body.Based on the image magnification of the microscope, a 200×200 pixelcropping was performed around the estimated center. This cropping kept atypical cell completely inside the frame. Images where two or more cellswere seen clumped together were discarded. Less than 5% of images showedsuch clumping behavior of cells. The number of pixels was optimized toincrease the speed as well as to retain the required information.

H. Contour Detection

After initial Wiener filtering, contrast enhancement and smoothing,separated cell image contours were detected using “level set” algorithm,as described in Sethian, J. A. (1996) Proceedings of the NationalAcademy of Sciences 93, 1591. Energy parameters were defined for eachimage and an initial contour was estimated. A recursive algorithm wasdeveloped to minimize the total energy in the equation. Minimum energyoccurred at the cell membrane of the image where change in contrast frompixel to pixel became the largest. Contour image plot was then convertedto binary format for further analysis. Binary morphological imageprocessing functions ‘erode’ and ‘dilate’ were used to eliminatespurious pixels. The cellular region in the binary image was representedas white and the rest of the frame was represented as black (FIG. 33).This conversion made it suitable to statistically analyze the extracteddata, without losing any important morphological information.

I. Feature Extraction

Centroids for all cells were determined by taking average pixel count inboth horizontal and vertical directions. Cell membrane distance from thecenter was calculated at a resolution of 24 degrees (FIG. 34). A totalof 15 radii were calculated for each cell. Resolution was optimized tomaximize the differences in the radii. Too low a number would havemissed important data, whereas more number of radii would have failed toamplify the differences between successive radii.

Cancer cells showed random shape changes after capture on the surface.The shapes changed from oval to elliptical, and then highly non-uniformshapes with multiple pseudopods emerged. The shape randomness wastracked from frame to frame for each cell. Non-uniformity of cells wascalculated from the differential of two successive radii. For twosuccessive radii, r_(n) and r_(n+1), the differential (Δr) wasΔr=r_(n)−r_(n+1). An empirical threshold was determined to amplify thedifferential (where the amplified differential is Δr′), and anon-uniformity parameter (N.U.) was then calculated. These steps aredescribed by equations (1) to (3) below:

$\begin{matrix}{{{\Delta\; r_{n}} = {r_{n + 1} - r_{n}}};} & (1) \\{{{\Delta\; r^{\prime}} = \begin{Bmatrix}0 & {{{if}\mspace{14mu}\left( {\Delta\; r} \right)} < {9\mspace{14mu}{pixels}}} \\\left( {\Delta\; r} \right)^{2} & {{{if}\mspace{14mu}\left( {\Delta\; r} \right)} > {9\mspace{14mu}{pixels}}}\end{Bmatrix}};{and}} & (2) \\{{{non}\text{-}{uniformity}\mspace{14mu}\left( {N.U} \right)} = {\sum\limits_{n = 1}^{N - 1}\;{\left( {\Delta\; r^{\prime}} \right).}}} & (3)\end{matrix}$

“Hausdorff distance” is a standard measure to determine the variationfrom one image to another by calculating and comparing point-to-pointdistance between contours. Hausdorff distances between cells in twoconsecutive frames were thus measured based on pixel level information,as described in Huttenlocher et al. (1993) “Pattern Analysis and MachineIntelligence,” IEEE Transactions on 15, 850-63. This approach calculatedthe maximum value among all minimum distances between any two possiblepoint sets on the two cell membranes. A comparison was made frame byframe for healthy and cancerous cells.

Pseudopods were computationally defined as an extension of the membraneover a threshold multiplier of the average radius. Such extensions werecalculated and tracked for a 360 degree rotation of radius for everyframe. Cancer cells showed random extension and contraction ofpseudopods, whereas normal cells remained still and did not show muchactivity. Hence change in number of pseudopods over time was animportant discriminating factor in this context. The rate of change innumber of pseudopods from frame to frame was thus calculated. Anextension was considered a positive change and its contraction wascounted as negative. Formation of pseudopods at different angles wasmeasured and recorded to keep track of the cell wall changes.

Results and Discussion

As described above, the RNA aptamers on the surface created a passivemonolayer that inhibited regular cell-surface interaction throughadhesion molecules. On bare glass, astrocytes indeed interact with thesurface, though at a much reduced pace. A comparison of astrocytescell-surface interaction on a piranha cleaned bare glass and a RNAfunctionalized glass substrate is presented in FIG. 35.

However, such surfaces may introduce non-specific cell activity andfalse detection. Because of the RNA monolayer, while the other modes ofcell-surface interaction are disabled, only the complementary moleculeson the cell membrane can possibly interact with the surface. Hence itcan be important to ensure a uniform and dense monolayer to inhibitnon-specific interaction. Binding of probe DNA and RNA to the substratewas verified through staining by acridine orange.

In all cases, before incubating on the substrates, cells werecentrifuged and the supernatants were removed. Sterilized 1×PBS solution(with 5 mmol/L MgCl₂) was added to dilute the cells. The functionalizedslides were placed in PDMS wells and 0.5 ml cell solution was placed oneach substrate to make sure it was completely submerged. After 3-4minutes of settling, images were captured at 30 seconds interval. Theperiod was optimized to capture maximum activity and to minimizeprocessing overhead. Tumor cells on the aptamer modified surfaces showedenhanced activities (shape changes with time, non-uniformity, andformation of pseudopods etc.) while attaching to the surface. All fourkinds of samples (two anti-EGFR modified surfaces and two surfacesmodified with mutant aptamers) were measured on the same day. The cellswere taken out of the incubator and kept at 37° C. The experiment wasdone both inside and outside an incubation chamber, and negligibledifferences were observed during the short time of imaging. However,complete data acquisition process was done within 20 minutes to minimizeartifacts from unwanted cell deaths.

After image acquisition, each cell image was separated out and contourswere detected. On average, the time for such contour detection dependedon a number of factors such as the microscope light and aperture size.These factors were optimized to enhance the edge contrast of the cellswhich resulted in more efficient and faster processing speed, as a lowernumber of computational iterations were needed. The algorithm could betuned for computing either smoother surfaces of the binary images orfaster processing speed. For rapid processing, the edges were keptslightly rough. The roughness eventually averaged out over the wholemembrane and minimally affected the extracted data.

During the processing, most errors occurred due to limitations on thedepth of field of the imaging microscope. When a pseudopod extended, theboundary of the pseudopod region went slightly out of focus and thiscreated some distortions. Sequential images of 100 regions, eachmeasuring the changes in the cell contours over time, were taken on amoving-stage microscope to ensure cell activity was recorded across thewhole surface of the chip. The analysis of the 100 regions showedstatistically different behavior between cancer and normal cells.

There were four combinations of cells and functionalized surfaces: (i)hGBM cells+anti-EGFR aptamer surface, (ii) Astrocytes+anti-EGFR aptamersurface, (iii) hGBM cells+mutant aptamer surface and (iv)Astrocytes+mutant aptamer surface. The last three combinations acted ascontrols for the experiments. Every cell in a frame was tracked andunderwent image processing and data analysis. Non-uniformities andHausdorff distances for the four combinations were calculated.

The hGBM cells on anti-EGFR aptamer surface showed much highernonuniformity in their surface contour over the period of imageacquisition. The same cells remained inactive on a non EGFR-specificmutant aptamer coated surface (FIG. 36). In addition, as describedabove, the control cells (astrocytes) also do not show such activity ormorphological changes on the EGFR-specific surface. Enhanced activity ofthe cancer cells on the surface resulted in higher cell nonuniformity.Over 25 frames, tumor cells showed non-uniformity ranging from 8-10(a.u.) on average, whereas in the controls for other combinations, thisremained below 1 (a.u.), thus providing a non-uniformity factor ofgreater than 8-10 (where the “non-uniformity factor” is the ratio of thenon-uniformity of the tumor cells compared to the non-uniformity of thecontrol cells when measured as described herein). In some cases, thenon-uniformity factor is about 2-100, 2-50, 2-20, 5-100, 5-50, 5-20,5-10, 10-100, 10-50, or 10-20.

To verify whether such activity was EGFR specific, in a separateexperiment, astrocytes cells were incubated on both the anti-EGFR andthe mutant aptamer coated control surfaces. These cells showednegligible activity on the surfaces (FIG. 37). This result indicatesthat the surface was indeed passivated by the RNA layer and acomplementary ligand on the surface can activate the cell towards shapechanges. Such activity might could also be triggered by a complementaryantibody layer on the surface.

The Hausdorff distance between the contours of cells also showeddistinguishing behavior, as illustrated in FIG. 38. The tumor cells onthe anti-EGFR aptamer surfaces consistently showed higher Hausdorffdistances compared to the controls. On average, the Hausdorff distancewas calculated to be 4500 (a.u.) for tumor cells whereas for controlcombinations, it stayed around 200 (a.u.), thus providing a Hausdorffdistance factor of about 22.5 (where the “Hausdorff distance factor” isthe ratio of the Hausdorff distance of the tumor cells compared to theHausdorff distance of the control cells when measured as describedherein). In some cases, the Hausdorff distance factor is about 2-100,2-50, 2-40, 5-100, 5-50, 5-30, 10-100, 10-50, 10-40, 10-30, 20-100,20-50, 20-40, or 20-30.

The rates of change of pseudopods between frames are shown in TABLE V.On average, the tumor cells on anti-EGFR aptamer surface showedpseudopods forming at different locations on the wall of the same cellas seen in consecutive frames. There was constant formation andcontraction of pseudopods. Each contraction was considered as a changeof −1 while formation of a new pseudopod was counted as +1. On the otherhand, control combinations showed minimal changes in cell contour. Evenif there were pseudopods at start, these stayed at the same orientationin all subsequent frames.

TABLE V Differential Change over 25 frames Cell and Data set no. surfacecombination 1 2 3 4 5 6 7 8 9 10 Astrocytes on anti-EGFR 0 0 0 0 5 3 2 50 1 aptamer Astrocytes on mutant 0 0 3 0 0 4 0 0 1 0 aptamer hGBM onanti-EGFR 20 7 28 12 6 11 9 7 5 14 aptamer hGBM on mutant 0 0 1 0 0 0 00 1 0 aptamer

Cell motility is a natural phenomenon, where cells move by protruding asection of the membrane. This complex process is accomplished bysophisticated force balancing acts between internal cytoskeletonstructures, adhesion molecules and the cell membrane. Cytoskeletonstructures works in coordination of actin filament, microtubules, andtubulin protein. Major force contribution comes from the actin filamentthat polymerizes and thus changes length in response to physical andchemical stimuli. Depending on the actin concentration on the membrane,several nano-Newtons of force on the membrane is applied by the actinfilament structure. This force is calculated from theassociation/disassociation constant of the actin monomer to thefilament. On the other hand, the membrane molecules are dynamic innature due to Brownian motion and are flexible when subjected toexternal force. Different models such as ratchet models andautocatalytic models have been proposed to describe molecular mechanismsof actin force generation and protrusion of the leading edge.

Flexibility of the cancer cell membranes are reported to be higher thanthe healthy cells due to their inherent weak structures. Because ofoverexpression of several proteins on the membrane of cancer cells, thebalancing forces between the cytoskeleton and the membrane protein aredifferent than healthy cells. The hGBM cells, with strong overexpressionof the EGFR, when seeded on a surface functionalized with anti-EGFRaptamer have an added parameter in the force balancing equation. Thesurface passivation due to anti-EGFR aptamer coating on the surfacereduces adhesion while the binding interaction between EGFR and antiEGFR aptamer interaction enhances the membrane protrusion. This leads toenhanced cell movement activity. However, having no certain directionalsignaling, cells do not show significant translational movement.

Conclusion

The foregoing Example illustrates that quantitative differences in theinteractions of normal and tumor cells on functionalized surfaces can beused as a rapid cytological indicator of cancer cells. By usingappropriate image processing techniques in combination with suitablesurface preparation, a detection method for tumor cells can beimplemented. Thus, methods described herein, in some embodiments, canserve as an additional modality to support histological findings and/orto identify tumor cells based on their physical behavior from bloodsamples or biopsy specimen drawn directly from patients.

Example 6

In this Example, a method of differentiating tumor cells from non-tumorcells was carried out in a manner similar to that described above inExample 5.

Materials and Methods

Nanotextured and plain glass slides were used for cancer cell isolation.In either case, the surfaces were functionalized with anti-EGFR aptamerthrough multi-step layer by layer assembly of molecules, as describedabove. First, the slides were cleaned with Piranha solution (H2O2:H2SO4,1:1) followed by silanization. A single-stranded DNA (ssDNA) sequencewas then attached to the surfaces through a homo-bifunctional linker.The linker would bind to the silane on one end and the amine group ofssDNA on the other end. After ssDNA attachment and blocking ofunreactive linker functional groups, anti-EGFR RNA was hybridized to thessDNA on the chips. After incubating the chips with tumor cell mixtures,time lapsed images were taken.

These recorded images were then processed though several steps towardsfinal contour detection. After initial image enhancement, cellboundaries were detected and images were converted to binary format forprocessing. The temporal changes in contours of cells from frame toframe gave information like size, the change in cell boundary, thegrowth, and the formation of pseudopods. Based on these features severalfeature vectors were defined that would differentiate one cell type fromthe other, as described further herein.

The image enhancement operations showed dependence of the computationspeed on a number of factors (e.g., the density of cells in a frame, thefrequency of frame capture by the camera, the depth of field of theimaging plane, the resolution of the captured image, and the ability toreimage the same cell (or set of cells) at exactly same position). Theprocesses consisted of tone detection (dark versus bright areas),identification of bright region (cell tagged), segmentation (conversionto binary image), angular sectoring (to breakdown circular cell intoparts), boundary tracing (cell growth and motility) and detection ofprotrusions (pseuodpods).

Results

EGFR-specific aptamers selectively isolated EGFR overexpressing humanglioblastoma (hGBM) cells with high specificity. Tumor cells, when boundto such functionalized surfaces, showed distinct morphological patternsand enhanced activity as compared to healthy cells, which remained calm.These cells showed clear changes in cell shapes from spherical tosemi-elliptical with very flat orientation, formed pseudopods (possiblyto cover much more surface area) and showed rapid growth. Cell behavioron both EGFR-specific surface and control surface was quantified anddifferent feature vectors like non-uniformity (FIG. 39), Hausdorffdistance, frame-to-frame differential pseudopod formations werecalculated. The non-uniformity feature was based on the formation offinger-like projections protruding out of the cell walls. This also madethe focal point move and required manual focusing of the microscope. Afrequency of 4 images per minute was found to be optimal to essentiallycapture the slow movements of the cells.

That which is claimed is:
 1. A method of detecting tumor cellscomprising: providing a device, the device comprising: a substratesurface; and a plurality of first aptamer probes attached to thesubstrate surface; contacting a plurality of cells with the plurality offirst aptamer probes attached to the substrate surface; adhering one ormore of the plurality of cells to the substrate surface; and measuring aHausdorff distance of at least one adhered cell.
 2. The method of claim1 further comprising using the Hausdorff distance of the adhered cell toidentify the adhered cell as a tumor cell or a non-tumor cell.
 3. Themethod of claim 1, wherein the substrate surface is formed from glass,silicon, or a polymer.
 4. The method of claim 1, wherein the pluralityof first aptamer probes forms a uniform or substantially uniform coatingon the substrate surface.
 5. The method of claim 4, wherein at leastabout 80% of the substrate surface is coated by the plurality of firstaptamer probes.
 6. The method of claim 1, wherein the plurality of firstaptamer probes comprises an anti-EGFR aptamer.
 7. The method of claim 1,wherein the plurality of cells comprises tumor cells.
 8. The method ofclaim 7, wherein the tumor cells comprise circulating tumor cells. 9.The method of claim 7, wherein the tumor cells comprise breast cancercells, cervical cancer cells, lung cancer cells, bladder cancer cells,ovarian cancer cells, esophageal cancer cells, head and neck cancercells, kidney cancer cells, glioma cells, bladder cancer cells,pancreatic cancer cells, colon cancer cells, or a combination thereof.10. The method of claim 1, wherein the plurality of cells comprises amixture of tumor cells and non-tumor cells.
 11. The method of claim 1,wherein contacting a plurality of cells with the plurality of firstaptamer probes comprises contacting a bodily fluid with the plurality offirst aptamer probes.
 12. The method of claim 11, wherein the bodilyfluid comprises blood, saliva, urine, or bladder wash.
 13. The method ofclaim 10, wherein more than one cell is adhered to the substrate surfaceand the adhered cells exhibit a Hausdorff distance factor of 2-100, theHausdorff distance factor being a ratio of the Hausdorff distance of theadhered tumor cells compared to the Hausdorff distance of the adherednon-tumor cells.
 14. The method of claim 1, wherein the substrate isformed from silicon.
 15. A method of detecting tumor cells comprising:providing a device, the device comprising: a substrate surface; and aplurality of first aptamer probes attached to the substrate surface;contacting a plurality of cells with the plurality of first aptamerprobes attached to the substrate surface; adhering one or more of theplurality of cells to the substrate surface; and measuring a change inthe number of pseudopods of at least one adhered cell.
 16. The method ofclaim 15, further comprising using the change in the number ofpseudopods of the adhered cell to identify the adhered cell as a tumorcell or a non-tumor cell.
 17. The method of claim 15, wherein thesubstrate surface is formed from glass, silicon, or a polymer.
 18. Themethod of claim 15, wherein the plurality of first aptamer probes formsa uniform or substantially uniform coating on the substrate surface. 19.The method of claim 18, wherein at least about 80% of the substratesurface is coated by the plurality of first aptamer probes.
 20. Themethod of claim 15, wherein the plurality of first aptamer probescomprises an anti-EGFR aptamer.